Alteration and modulation of protein activity by varying post-translational modification

ABSTRACT

Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

RELATED APPLICATIONS

The present application claims priority as a continuation-in-part under35 U.S.C. §119 to the following: U.S. patent application Ser. No.12/380,450 (filed Feb. 26, 2009. This applicant also claims priority asa continuation-in-part under 35 U.S.C. §119 to the following: U.S.patent application Ser. No. 11/266,063 (filed Nov. 2, 2005). Theentirety of each of which is incorporated herein by this reference.

GOVERNMENT RIGHTS

This invention was made with government support under Contract NumbersDE-AC07-99ID13727 and DE-AC07-05ID14517 awarded by the United StatesDepartment of Energy. The government has certain rights in theinvention.

STATEMENT ACCORDING TO 37 C.F.R. §1.52(e)(5)—SEQUENCE LISTING SUBMITTEDON COMPACT DISC

Pursuant to 37 C.F.R. §1.52(e)(1)(ii), a compact disc containing anelectronic version of the Sequence Listing has been submittedconcomitant with this application, the contents of which are herebyincorporated by reference. A second compact disc is submitted and is anidentical copy of the first compact disc. The discs are labeled “copy 1”and “copy 2,” respectively, and each disc contains one file entitled“Seq list BA 315.txt” which is 964 KB and created on Dec. 16, 2009.

TECHNICAL FIELD

The present invention relates generally to the field of biotechnology.More specifically, embodiments of the present invention relate topost-translational modification of proteins.

BACKGROUND

It has been believed until only very recently that bacteria in generaldo not glycosylate their proteins. While there have been some instancesreported, these were dismissed as unusual anomalies (Borman 2006). It isnow becoming more accepted that bacteria do glycosylate their proteinsin perhaps more ways than eukaryotes do, although this belief is not yetwidespread (Schäffer et al., 2001). In a recent review article, it wasstated that glycosylation has been shown to assist in protein stability,modulate physical properties such as solubility, protect againstproteolysis, modify activity profiles, and target for externalization(Upreti et al., 2003). In 1994, a group purified an amylase fromAlicyclobacillus acidocaldarius and showed that the amylase wascell-bound during active growth (Schwermann et al., 1994). As theculture entered stationary phase, the cells released an active solubleglycosylated version of the amylase into the medium (Schwermann et al.,1994). No attempt was made to compare the activities of the variousforms of the amylases.

BRIEF SUMMARY

Embodiments of the invention include methods of altering the enzymaticactivity of an extremophilic enzyme or other protein via means ofchemical glycosylation and/or isolated or partially purifiedglycosyltransferases and/or post-translational modification proteins,extracts of cells comprising glycosyltransferases and/orpost-translational modification proteins, and/or in cells comprising oneor more glycosyltransferases and/or post-translational modificationproteins. Embodiments of the invention include methods ofpost-translationally modifying proteins. In some embodiments, thepost-translational modification may occur using means of glycosylation(including chemical glycosylation), pegylation, phosphorylation,methylation or other forms of post-translational modification and/or beisolated or partially purified glycosyltransferases and/orpost-translational modification proteins, extracts of cells comprisingglycosyltransferases and/or post-translational modification proteins,and/or in cells comprising one or more glycosyltransferases and/orpost-translational modification proteins. Embodiments of the inventioninclude post-translationally modified proteins including, but notlimited to, SEQ ID NOS:307 (celB), 331 (an Endoglucanase C), 333 (aPeptidoglycan N-acetylglucosamine deacetylase), 335 (aBeta-galactosidase), 337 (an arabinofuranosidase), and 338 (analpha-xylosidase). Embodiments thus include glycosylated versions of theaforementioned proteins.

A first aspect of the present invention relates to an enzyme isolatedfrom an extremophilic microbe that displays optimum enzymatic activityat a temperature of greater than about 80° C., and a pH of less thanabout 2.

Another aspect of the present invention relates to a hemicellulase thatwas derived from Alicyclobacillus acidocaldarius (ATCC 27009).

Another aspect of the present invention relates to an enzyme that isuseful in the degradation of complex biomolecules.

Still further, another aspect of the present invention relates to anenzyme that may be useful in a simultaneous saccharification andfermentation process to convert a biomass sugar into an end product.

Yet another aspect of the present invention relates to a method for thetreatment of a biomass that includes the steps of providing a source ofa biomass having a biomass sugar; pretreating the biomass with awater-soluble hemicellulase derived from Alicyclobacillus acidocaldarius(ATCC 27009) to produce an end product.

Another aspect of the present invention relates to a method for thepreparation of a hemicellulase that includes the steps of providing asource of Alicyclobacillus acidocaldarius (ATCC 27009); cultivating theAlicyclobacillus acidocaldarius (ATCC 27009) in a microbial nutrientmedium having a supernatant; separating the cells of theAlicyclobacillus acidocaldarius from the nutrient medium supernatant;and recovering and purifying the hemicellulase derived from theAlicyclobacillus acidocaldarius (ATCC 27009) from the nutrient mediumsupernatant.

Moreover, another aspect of present invention relates to a method forhydrolyzing a polysaccharide that includes the steps of providing awater-soluble hemicellulase derived from a microbe; and conductinghydrolysis of a polysaccharide with the water-soluble hemicellulase at apH of less than about 2.

These and other aspects of the present invention will be described ingreater detail hereinafter.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 depicts a sequence alignment between SEQ ID NO:1 (RAAC00164) andref|YP_(—)001223775.1|, ref|YP_(—)729290.1|, ref|ZP_(—)01084440.1|,ref|ZP_(—)01079150.1|, and ref|ZP_(—)01471594.1| (SEQ ID NOS:3-7)respectively, which all have the function assigned to SEQ ID NO:1 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 2 depicts a sequence alignment between SEQ ID NO:18 (RAAC00517) andref|ZP_(—)00589533.1|, ref|ZP_(—)01386435.1|, ref|YP_(—)378533.1|,ref|ZP_(—)00513158.1|, and ref|YP_(—)374173.1| (SEQ ID NOS:20-24)respectively, which all have the function assigned to SEQ ID NO:18 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 3 depicts a sequence alignment between SEQ ID NO:35 (RAAC00650) andref|YP_(—)001127183.1|, ref|ZP_(—)02038504.1|, ref|YP_(—)001647987.1|,ref|YP_(—)001377114.1|, and ref|NP_(—)835081.1| (SEQ ID NOS:37-41)respectively, which all have the function assigned to SEQ ID NO:35 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 4 depicts a sequence alignment between SEQ ID NO:52 (RAAC00991) andref|ZP_(—)02327412.1|, ref|YP_(—)001487207.1|, ref|ZP_(—)01172765.1|,ref|NP_(—)831314.1|, and ref|NP_(—)844008.1| (SEQ ID NOS:54-58)respectively, which all have the function assigned to SEQ ID NO:52 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 5A and 5B depict a sequence alignment between SEQ ID NO:69(RAAC01110) and ref|YP_(—)001519856.1|, ref|YP_(—)711688.1|,ref|ZP_(—)01331931.1|, ref|YP_(—)001076955.1|, and ref|YP 336440.1| (SEQID NOS:71-75) respectively, which all have the function assigned to SEQID NO:69 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 6A and 6B depict a sequence alignment between SEQ ID NO:86(RAAC01166) and gb|AAR99615.1|, gb|ABM68334.2|, ref|ZP_(—)01372248.1|,ref|YP_(—)519555.1|, and ref|ZP_(—)02234077.1| (SEQ ID NOS:88-92)respectively, which all have the function assigned to SEQ ID NO:86 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 7 depicts a sequence alignment between SEQ ID NO:103 (RAAC01167)and ref|ZP_(—)01515212.1|, ref|YP_(—)001277643.1|,ref|ZP_(—)02291400.1|, ref|YP_(—)001633727.1|, andref|YP_(—)001434357.1| (SEQ ID NOS:105-109) respectively, which all havethe function assigned to SEQ ID NO:103 in Table 1. Amino acids conservedamong all sequences are indicted by a “*” and generally conserved aminoacids are indicated by a “:”.

FIGS. 8A and 8B depict a sequence alignment between SEQ ID NO:120(RAAC01170) and ref|YP_(—)001324592.1|, ref|YP_(—)342776.1|,ref|NP_(—)780975.1|, ref|YP_(—)001636830.1|, and ref|YP_(—)001299026.1|(SEQ ID NOS:122-126) respectively, which all have the function assignedto SEQ ID NO:120 in Table 1. Amino acids conserved among all sequencesare indicted by a “*” and generally conserved amino acids are indicatedby a “:”.

FIG. 9 depicts a sequence alignment between SEQ ID NO:137 (RAAC01248)and ref|ZP_(—)02170160.1|, ref|ZP_(—)01171895.1|, ref|YP_(—)076646.1|,ref|YP_(—)590910.1|, and ref|ZP_(—)02175410.1| (SEQ ID NOS:139-143)respectively, which all have the function assigned to SEQ ID NO:137 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 10A and 10B depict a sequence alignment between SEQ ID NO:154(RAAC01348) and ref|ZP_(—)01665289.1|, ref|ZP_(—)01643350.1|,gb|AAW77167.1|, ref|YP_(—)452722.1|, and ref|ZP_(—)02241787.1| (SEQ IDNOS:156-160) respectively, which all have the function assigned to SEQID NO:154 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIG. 11 depicts a sequence alignment between SEQ ID NO:171 (RAAC01377)and ref|YP_(—)147952.1|, ref|YP_(—)520670.1|, ref|YP_(—)001395809.1|,ref|YP_(—)001309701.1|, and ref|YP_(—)001643660.1| (SEQ ID NOS:173-177)respectively, which all have the function assigned to SEQ ID NO:171 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 12 depicts a sequence alignment between SEQ ID NO:188 (RAAC01611)and ref|YP_(—)146214.1|, ref|YP_(—)001124463.1|, ref|NP_(—)865262.1|,ref|YP_(—)426013.1|, and ref|ZP_(—)01885526.1| (SEQ ID NOS:190-194)respectively, which all have the function assigned to SEQ ID NO:188 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 13A and 13B depict a sequence alignment between SEQ ID NO:205(RAAC01612) and ref|YP_(—)146215.1|, ref|YP_(—)001124464.1|,ref|YP_(—)074948.1|, ref|YP_(—)001039503.1|, and ref|NP_(—)621770.1|(SEQ ID NOS:207-211) respectively, which all have the function assignedto SEQ ID NO:205 in Table 1. Amino acids conserved among all sequencesare indicted by a “*” and generally conserved amino acids are indicatedby a “:”.

FIGS. 14A and 14B depict a sequence alignment between SEQ ID NO:222(RAAC01926) and ref|YP_(—)001038202.1|, ref|ZP_(—)01667587.1|,ref|ZP_(—)01575301.1|, ref|YP_(—)001211020.1|, and ref|Y_(—)516465.1|(SEQ ID NOS:224-228) respectively, which all have the function assignedto SEQ ID NO:222 in Table 1. Amino acids conserved among all sequencesare indicted by a “*” and generally conserved amino acids are indicatedby a “:”.

FIG. 15 depicts a sequence alignment between SEQ ID NO:239 (RAAC01998)and ref|NP_(—)348940.1|, ref|NP_(—)721244.1|, dbj|BAC75700.1|,ref|ZP_(—)00605123.1|, and ref|YP_(—)015329.1| (SEQ ID NOS:241-245)respectively, which all have the function assigned to SEQ ID NO:239 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 16 depicts a sequence alignment between SEQ ID NO:256 (RAAC02011)and ref|YP_(—)754819.1|, ref|YP_(—)184322.1|, ref|NP_(—)577787.1|,ref|NP_(—)142068.1|, and ref|NP_(—)125751.1| (SEQ ID NOS:258-262)respectively, which all have the function assigned to SEQ ID NO:256 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 17A and 17B depict a sequence alignment between SEQ ID NO:273(RAAC02381) and ref|NP_(—)622177.1|, ref|YP_(—)848858.1|,ref|YP_(—)001374688.1|, ref|NP_(—)470039.1|, and ref|ZP_(—)01929325.1|(SEQ ID NOS:275-279) respectively, which all have the function assignedto SEQ ID NO:273 in Table 1. Amino acids conserved among all sequencesare indicted by a “*” and generally conserved amino acids are indicatedby a “:”.

FIG. 18 depicts a sequence alignment between SEQ ID NO:290 (RAAC02421)and ref|ZP_(—)01721811.1|, ref|NP_(—)241897.1|, ref|YP_(—)001486101.1|,ref|ZP_(—)01170532.1|, and ref|ZP_(—)02327994.1| (SEQ ID NOS:292-296)respectively, which all have the function assigned to SEQ ID NO:290 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 19 is a graph depicting an effect of temperature on xylanaseactivity, as provided by the present invention.

FIG. 20 is a graph depicting an effect of temperature on cellulaseactivity, as provided by the present invention at a pH of 4.0.

FIG. 21 is a graph depicting an effect of pH on cellulase activity ofthe present invention at a temperature of 60° C.

FIG. 22 is a graph depicting an effect of pH on xylanase activity of thepresent invention at a temperature of 60° C.

FIG. 23 is a graph depicting xylanase activity of SEQ ID NO:307 asdetermined using Wheat arabinoxylan (WAX) at various pH and temperaturelevels. The enzyme was isolated from Alicyclobacillus acidocaldarius(black bars) or produced in E. coli (white bars). There is no availabledata for enzyme isolated from Alicyclobacillus acidocaldarius at pH 5.5(60° C. and 80° C.).

FIG. 24 is a graph depicting cellulase activity of SEQ ID NO:307 asdetermined using carboxymethyl cellulose (CMC) at various pH andtemperature levels. The enzyme was isolated from Alicyclobacillusacidocaldarius (black bars) or produced in E. coli (white bars). Thereis no available data for enzyme isolated from Alicyclobacillusacidocaldarius at pH 5.5 (60° C. and 80° C.).

FIG. 25 is a graph depicting a ratio of cellulose/xylanase activity ofSEQ ID NO:307 as determined from FIGS. 23 and 24 at various pH andtemperature levels. The enzyme was isolated from Alicyclobacillusacidocaldarius (black bars) or produced in E. coli (white bars). Thereis no available data for enzyme isolated from Alicyclobacillusacidocaldarius at pH 5.5 (60° C. and 80° C.). The tops of the bars at pH5.5 for the enzyme produced in E. coli are left open to indicate thatthe ratio was greater than 10.

FIG. 26 is a graph depicting xylanase activity of SEQ ID NO:307 asdetermined using wheat arabinoxylan (WAX) at various pH and temperaturelevels. The enzyme was produced in Pichia pastoris (black bars) orproduced in E. coli (white bars).

FIG. 27 is a graph depicting cellulase activity of SEQ ID NO:307 asdetermined using carboxymethyl cellulose (CMC) at various pH andtemperature levels. The enzyme was produced in Pichia pastoris (blackbars) or produced in E. coli (white bars).

FIG. 28 is a graph depicting a ratio of cellulose/xylanase activity ofSEQ ID NO:307 as determined from FIGS. 26 and 27 at various pH andtemperature levels. The enzyme was produced in Pichia pastoris (blackbars) or produced in E. coli (white bars). The tops of the bars at pH5.5 for the enzyme produced in E. coli are left open to indicate thatthe ratio was greater than 10.

FIG. 29 is a graph depicting xylanase activity of SEQ ID NO:307 asdetermined using wheat arabinoxylan (WAX) at various pH and temperaturelevels. The enzyme was isolated from Alicyclobacillus acidocaldarius(black bars) or produced in Pichia pastoris (white bars). There is noavailable data for enzyme isolated from Alicyclobacillus acidocaldariusat pH 5.5 (60° C. and 80° C.).

FIG. 30 is a graph depicting cellulase activity of SEQ ID NO:307 asdetermined using carboxymethyl cellulose (CMC) at various pH andtemperature levels. The enzyme was isolated from Alicyclobacillusacidocaldarius (black bars) or produced in Pichia pastoris (white bars).There is no available data for enzyme isolated from Alicyclobacillusacidocaldarius at pH 5.5 (60° C. and 80° C.).

FIG. 31 is a graph depicting a ratio of cellulose/xylanase activity ofSEQ ID NO:307 as determined from FIGS. 29 and 30 at various pH andtemperature levels. The enzyme was isolated from Alicyclobacillusacidocaldarius (black bars) or produced in Pichia pastoris (white bars).There is no available data for enzyme isolated from Alicyclobacillusacidocaldarius at pH 5.5 (60° C. and 80° C.).

FIG. 32 is a graph depicting xylanase activity of SEQ ID NO:307 lackingthe C-terminal 203 amino acids as determined using wheat arabinoxylan(WAX) at various pH and temperature levels. The enzyme was produced inPichia pastoris (black bars) or produced in E. coli (white bars).

FIG. 33 is a graph depicting cellulase activity of SEQ ID NO:307 lackingthe C-terminal 203 amino acids as determined using carboxymethylcellulose (CMC) at various pH and temperature levels. The enzyme wasproduced in Pichia pastoris (black bars) or produced in E. coli with anN-terminal His tag (white bars).

FIG. 34 is a graph depicting a ratio of cellulose/xylanase activity ofSEQ ID NO:307 lacking the C-terminal 203 amino acids as determined fromFIGS. 32 and 33 at various pH and temperature levels. The enzyme wasproduced in Pichia pastoris (black bars) or produced in E. coli (whitebars).

FIG. 35 is a graph depicting a ratio of arabinofuranosidase activity ofRAAC00307 (SEQ ID NO:337) produced in E. coli. Activity at 50° C.(diamonds), 60° C. (squares), 70° C. (triangles), 80° C. (“x”s), and 90°C. (“*”s) at various pH levels are shown. The enzyme had no activity atpH 2.

FIG. 36 is a graph depicting a ratio of beta xylosidase activity ofRAAC00307 (SEQ ID NO:337) produced in E. coli. Activity at 50° C.(diamonds), 60° C. (squares), 70° C. (triangles), 80° C. (“x”s), and 90°C. (“*”s) at various pH levels are shown. The enzyme had no activity atpH 2.

FIG. 37 is a graph depicting a ratio of beta xylosidase activity ofRAAC00307 (SEQ ID NO:337) produced in Pichia pastoris. Activity at 60°C. (diamonds) and 80° C. (squares), at various pH levels are shown.

DETAILED DESCRIPTION

One aspect of the present invention, as described hereinafter, relates,in part, to enzymes isolated from an extremophilic microbe that displayoptimum enzymatic activity at a temperature of greater than about 80°C., and an optimum pH of less than about 2. In further aspects of thepresent invention, the enzyme may be a hemicellulase and/or xylanasethat was derived from Alicyclobacillus acidocaldarius, where theorganism is further identified as ATCC 27009. The enzyme, as discussedhereinafter, appears to display enzymatic activity at a pH of about 1.Still further, this same enzyme has a molecular weight of at least about120 kDa. In the present invention, the enzyme, as disclosed, may beuseful in a simultaneous saccharification and fermentation processand/or a sequential hydrolysis and fermentation process to convert abiomass sugar into an end product. Still further, the enzyme, asdescribed herein, may be useful in the pretreatment of a biomass slurryto degrade a water-soluble or water-insoluble oligomer and/orpolysaccharide that is present in the biomass slurry to produce an endproduct.

As used hereinafter, the term “extremophilic microbe” means an organismthat can live and thrive under conditions that humans would considerextreme, such as boiling water, ice, battery acid or at the bottom ofthe ocean. Examples of such microbes include, but are not limited to,Pyrolobus fumarii that grows at temperatures up to 235° F. (113° C.),Psychrobacter cryopegella that survives at temperatures of −20° C. (−4°F.), Deinococcus radiodurans that can survive in a nuclear reactor,Photobacterium profundum that thrives at pressures 300 times theatmospheric pressure at sea level, and Picrophilus torridus that livesat a pH of 0, the same as battery acid. Environments in which thesemicrobes can be found include boiling hot springs, deep ocean thermalvents, glaciers, salt flats, and nuclear reactors. The microbes used inthe present invention can be obtained from natural and artificialsources or commercially from culture depositories. In the presentinvention, the cultivation is preferably conducted at temperatures above40° C. and a pH below about 5, and more preferably above 50° C. andbelow a pH of 4, and most preferably above 55° C. and below a pH of 3.5,and under anaerobic, aerobic, and/or micro-aerophilic conditions. Whilethe cultivation period varies depending upon the pH, temperature andnutrient medium used, a period of 12 hours to several days willgenerally give favorable results. As used herein, Alicyclobacillusacidocaldarius is defined as a microorganism that can be obtained fromthe American Type Culture Collection (ATCC), Manassas, Va., and that isidentified as Alicyclobacillus acidocaldarius (ATCC 27009).

As used hereinafter, the phrase “enzymatic activity” means the reactionan enzyme causes to occur. Enzymes are proteins produced by all livingorganisms that mediate, cause and/or promote reactions that change achemical into another type of chemical without themselves being alteredor destroyed. In the context of the present application, the word“optimum,” when used in combination with the term “enzymatic activity,”means the most favorable conditions that allow the enzyme to work thebest and the fastest for a given end result. The optimum enzymaticactivity may be affected by conditions that include temperature, pH, andsalt concentrations.

As used hereinafter, the word “xylanase” means an enzyme that breaksapart hemicellulose by breaking the chemical bonds between the xylosesugars that make up the backbone of the hemicellulose molecule, or bybreaking bonds between xylose sugars in the hemicellulose side chains.

The word “polysaccharide” as used hereinafter shall mean a chain ofsugars (can be the same sugars or different sugars) that are linkedtogether by chemical bonds. Polysaccharides can consist of straightchains of these sugars with or without side chains. Examples ofpolysaccharides include starch, pectin, cellulose, and hemicellulose.

The word “hydrolysis” in the context of this present application shallmean a chemical reaction in which water reacts with a molecule andbreaks it into at least two pieces.

As used hereinafter, the phrase “biomass sugar” shall mean sugars thathave come from the breakdown of biomass components, such as celluloseand hemicellulose. Examples of biomass sugars include, but are notlimited to, saccharides, glucose, xylose, galactose, mannose, arabinose,as well as combinations, oligomers, and/or modified or substituted formsthereof.

The phrase “simultaneous saccharification and fermentation process”shall mean hereinafter a process for making a fuel or chemical such asethanol from a biomass that may or may not have been pretreated bychemical means, and where cellulase and/or hemicellulase enzyme(s) areused to break down biomass polysaccharides into sugars(saccharification); and the sugars are fermented by source(s) ofmicroorganism(s) into the product fuel or chemical (fermentation). Thesetwo processes occur at the same time, in the same reaction vessel(simultaneous).

The phrase “end product” as used in the present application shall meanhereinafter the chemical(s) that is/are produced by a chemical orenzymatic reaction. Examples of end products contemplated by the presentinvention include simpler saccharides and sugars (e.g., monomers,dimers, trimers, oligomers, etc.), alcohols, fuels, and/or otherproducts of an enzymatic reaction.

The word “biomass” in the context of the present invention shall meanplant and other lignocellulosic material such as corn stalks, wheatstraw, and wood by-products, such as sawdust and the like.

The phrase “pretreatment of a biomass slurry” shall mean, in the contextof the present application, the preparation of a biomass for itssubsequent conversion to fuels, such as ethanol. This pretreatmentincludes the steps of grinding the biomass to a powder or smallparticles, and adding water (this constitutes a slurry). This slurry isthen treated by a number of methods designed to partially or completelyremove the lignin from the biomass, and convert the hemicellulose andcellulose into a form that can be more easily degraded into theircomponent sugars using enzymes such as cellulases and hemicellulases.Some pretreatments degrade hemicellulose to its component sugars whileleaving the cellulose as part of the solid residue. This treatment stepis called a “pretreatment” because it occurs before both the enzymaticdegradation step and before the fermentation step that converts thesugars into ethanol.

The phrase “water-soluble” in the context of the present invention shallmean a chemical or other substance that can be dissolved completely inwater without leaving any solid residue.

The word “hemicellulose” in the context of the present invention meansone component of a plant (the other two being cellulose and lignin),that is made of a linear chain of sugars such as xylose, or mannose thatare connected by a chemical bond. This linear chain also has branchesconsisting of sugars and other chemicals along the chain.

The word “hemicellulase” in the context of the present invention means aclass of enzymes that can break hemicellulose into its component sugarsand other chemical monomers. Examples of hemicellulases include, but arenot limited to, xylanases, mannanases, glucuronidases, andarabinofuranosidases.

The phrase “sequential hydrolysis and fermentation process” in thecontext of the present invention shall mean a process for making a fuelor chemical from the biomass, such as ethanol, and where the biomass istreated physically or with a reactive chemical or solvent, or mixturesthereof, to remove the lignin, and to convert the cellulose andhemicellulose present in the biomass into their component sugars or intoa form that can be more easily degraded into their component sugarsusing enzymes such as cellulases and hemicellulases. Examples of theseinclude grinding, milling, acids, alkalis, organosolvents, and the like.These treatments can be performed at temperatures ranging from ambientto 300° C. or more, and at pressures ranging from ambient to 2000 psigor more. The sugars, which are dissolved in water, are then cooled, andthe pH adjusted to neutral, and then subsequently fermented bymicroorganisms of various types into a product fuel(s) or chemical(s)(fermentation). These two processes occur in separate reaction vesselswith the hydrolysis step conducted first, and the fermentation stepconducted second (e.g., sequential).

The phrase “cultivating Alicyclobacillus acidocaldarius” in the contextof the present invention shall mean providing the aforementioned microbewith a food source (soluble or insoluble lignocellulose or other sourceof polysaccharides or sugars) and various vitamins and mineralsdissolved in water (this constitutes the nutrient medium), and givingthe microbe the proper conditions that allow it to grow (a temperatureof 140° F. (60° C.), a pH of 3.5, and oxygen).

The phrase “separating the cells of the Alicyclobacillus acidocaldarius”in the context of the present invention shall include means for removingthe bacterial cells from the nutrient medium by, for example,centrifugation.

The phrase “recovering and purifying the hemicellulase” in the contextof the present invention shall mean separating the hemicellulase enzymefrom the nutrient medium. In the present invention, a process calledcation exchange was used to separate hemicellulase from the nutrientmedium. In this regard, the nutrient medium (with hemicellulase) waspumped through the cation exchange material. When brought into contactwith the cation exchange material, the hemicellulase will attach itselfto the cation exchange material, but the nutrient medium will passthrough. The hemicellulase enzyme is then removed from the cationexchange material and is purified.

The phrase “microbial nutrient medium” in the context of the presentapplication means a food source for the microbe (Alicyclobacillusacidocaldarius) and vitamins and minerals, all dissolved in water andadjusted to the pH needed by the microbe to grow. More specifically, themicrobial nutrient medium includes about 1 gram per liter of Xylan;about 10 mM NH₄Cl; about 5.2 mM K₂HPO₄; about 0.8 mM MgSO_(4.7) H₂O;about 1.74 mM Na₂SO₄; about 25 mg per liter MgCl₂; about 6.6 mg perliter of CaCl₂; about 2.0 mg per liter MnSO₄; about 0.5 mg per literZnSO₄; about 0.5 mg per liter of boric acid; about 5 mg per liter ofFeCl₃; about 0.15 mg per liter of CuSO₄; about 0.025 mg per liter ofNaMoO₄; about 0.05 mg per liter of CoNO₃; about 0.02 mg per liter ofNiCl₂; about 0.08 mg per liter of pyridoxine hydrochloride; about 0.01mg per liter of folic acid; about 0.1 mg per liter of thiaminehydrochloride; about 0.04 mg per liter of riboflavin; about 0.08 mg perliter of nicotinamide; about 0.08 mg per liter of p-aminobenzoate; about0.01 mg per liter of biotin; about 0.0004 mg per liter cyanocobalamin;about 0.08 mg per liter D-pantothenic acid-Ca; about 0.02 mg per literof myo-inositol; about 0.05 mg per liter of choline bromide; about 0.02mg per liter of monosodium orotic acid; and about 0.1 mg per literspermidine, wherein the resulting nutrient medium is adjusted to a pH ofabout 3.5.

The word “supernatant” in the context of the preknt application shallmean the nutrient medium that is leftover after the bacterial cells aresubstantially removed from same.

The inventors have isolated and characterized temperature and acidstable endoglucanase and/or xylanases that demonstrate activity atelevated temperatures, and low pH, and that show stability whenincubated under these conditions for extended periods of time. Theinventors recognize that heat and acid stable hemicellulases andcellulases, as described hereinafter, have particular value in, or as anaccessory to, processes that would lead, on the one hand, to thereduction in the severity of pretreatment processes, earlier described,and/or the elimination of these limitations in various processes. Inthis regard, the inventors screened numerous organisms from YellowstoneNational Park and various culture collections for microbes that had theability to produce enzymes that were stable at both high temperature,and low pH. In this regard, water and sediment samples were collectedfrom six springs in the Norris Geyser Basin of Yellowstone NationalPark. These samples were inoculated into a liquid mineral salt mediumhaving a pH 3.5, and further containing either 0.5 grams per liter ofoat spelt xylan, or 0.5 grams per liter of ground corn cobs. Thesubsequent cultures were incubated at 80° C. and were observed daily forgrowth, both visually and microscopically. Still further, a search ofthe American Type Culture Collection (ATCC) and the Deutsche Sammlungvon Mikroorganismen Und Zellkulturen (DSMZ) yielded four possibleheterotrophic organisms whose optimal temperatures, and pH, for growth,were greater than about 60° C., and less than about a pH of 4. Theseseveral organisms were grown in the media recommended by ATCC or DSMZwith a carbon source replaced by either oat spelt xylan, or ground corncobs, as described above. These cultures where then later incubated attheir optimum growth temperature and a pH of 3.5. Subsequent microbialgrowth was assessed visually by the appearance of turbidity.

In this investigation, hemicellulase and/or cellulase activities werepresumptively assumed present if growth occurred in the presence ofxylan. Cultures where growth occurred were harvested after approximatelythree days incubation. Cells were removed from the culture bycentrifugation. The culture supernatant was concentrated at about1000-2000 fold using an AMICON® ultrafiltration cell with a 10000 MWCOmembrane. The subsequent supernatant concentrate was then tested forhemicellulase and cellulase activity using arsenomolybdate reducingsugar acid assay (previously described by Somogyi (1952), J. Biol. Chem.195:19-23) with wheat arabinoxylan (commercially secured from Megazyme),or carboxymethylcellulose (secured from Sigma-Aldrich). These were usedas substrates for the hemicellulase and cellulase activities,respectively. Standard conditions for the assays were set at 60° C., anda pH of about 3.5. As will be seen by reference to the drawings, thehemicellulase and cellulase activities were measured at temperatures upto about 90° C. to determine the optimum temperature for enzymaticactivity. The reducing sugar assay referenced above was modified bychanging the incubation temperature of the supernatant concentrate withthe substrate. Similarly, the enzyme activities were measured at a pHranging from 1 to about 8 to determine the optimum pH for the enzymeactivity. For these studies, the reducing sugar assay was modified bypreparing the assay components in the appropriate pH buffer (pH 1-2, 50mM sodium maleate or 50 mM glycine; pH 2-6, 50 mM sodium acetate; pH6-8, 50 mM sodium phosphate; and pH 8-9, 50 mM Tris).

In addition to the foregoing, stabilities of the hemicellulases andcellulases as a function of temperature and pH were examined byincubating the supernatant concentrate at a temperature of about 70° C.,and a pH of 2.0. In this regard, a layer of mineral oil was placed overthe concentrate to limit evaporation during this exam. Samples wereperiodically collected and assayed for hemicellulase and cellulaseactivity at the standard assayed conditions earlier described. Withrespect to the hemicellulase and cellulase reaction kinetics, these weredetermined using the reducing sugar assay with varying amounts of wheatarabinoxylan or carboxymethylcellulose. The reaction kinetics weredetermined at 60° C. and a pH of 3.5. Michaelis-Menten parameters Vmaxand Km were calculated by nonlinear analysis using ENZYME KINETICS PRO™that is available through SynexChem. After the process noted above, theinventors identified Alicyclobacillus acidocaldarius (ATCC 27009) forfurther examination.

Subsequently, a crude enzyme preparation was made by concentrating thecell free culture material. A subsequent SDS-page gel showed five majorbands and several minor bands. The subsequently calculated masses ofthese bands were consistent with other reported xylanases andcellulases. As seen in FIGS. 19, 20, 21, and 22, the inventorsdiscovered that the enzyme isolated from the Alicyclobacillusacidocaldarius, which is identified herein as ATCC 27009, had an optimumtemperature for enzymatic activity (xylanase and cellulase) at about 80°C. As seen in FIG. 19, the relative enzymatic activity is contrastedagainst the enzymatic activity that is provided by a similar enzyme thatis isolated from another similar microbe T. lanuginosus. Further, it wasfound that the isolated endoglucanase and/or xylanase exhibitedenzymatic activity at a pH as low as 1, with an optimum pH of 2, whilethe optimum pH for the cellulase activity was at a pH of about 4,although it did show some activity at a pH as low as 2. FIG. 22 showsthe xylanase activity as a function of pH as described above. To thebest knowledge of the inventors, the lowest optimum hemicellulase pHpreviously reported was in the reference to Collins (2005, FEMS, Micro.Review 29(1):3-23). It is conceivable that the present water-solubleendoglucanase and/or xylanase enzyme that has been isolated perhaps hasactivity at a pH lower than 1, however, presently, the reducing sugarassay reagents were unstable below a pH of 1. Further investigationrevealed that the newly isolated hemicellulase and cellulase activitiesshowed no decrease in activity when incubated at 70° C. and a pH of 2.The aforementioned investigation lead the inventors to conclude that theAlicyclobacillus acidocaldarius (ATCC 27009) is capable of growth on axylan substrate, and further produces extracellular hemicellulase andcellulase activity, which are both water-soluble and display significanthemicellulase activity at a pH of about 2, and which further has amolecular weight of at least about 120 kDa. Again, see FIG. 22.

The prior art discloses that an acid stable xylanase has been purifiedand characterized from Aspergillus kawachii that has a pH optimum of 2.0and a temperature optimum between 50° C. to 60° C. (Purification andProperties of Acid Stable Xylanases From Aspergillus kawachii, K. Ito,H. Ogasawara, T. Sugomoto, and T. Ishikawa, Bioscience, Biotechnologyand Biochemistry 56 (4):547-550, April 1992.) Additionally, severalxylanases have been reported with pH optima in the range of 4 to 5, andnumerous xylanases have been reported that have temperature optima up to100° C. However, in the inventors' knowledge, the enzyme as describedhereinafter, is the first enzyme known that has activities at such a lowpH, and at such a high temperature, as claimed herein. In addition tothe foregoing, the inventors are aware that a xylanase has been purifiedfrom the same organism, that is, Alicyclobacillus acidocaldarius (ATCC27009), that is reported to have xylanase activity associated with it.It is reported that this enzyme had a pH optimum of 4.0, and atemperature optimum of about 80° C. In this regard, a thermoacidophilicendoglucanase and/or xylanase (celB) from Alicyclobacillusacidocaldarius (ATCC 27009) displayed high sequence similarity toarabinofuranosidases belonging to Family 51 of glycoside hydrolases (K.Eckert and E. Schneider, European Journal of Biochemistry 270(17):3593-3602, September, 2003). The aforementioned cellulase precursoras described in this prior art reference is best understood by a studyof SEQ ID NO:307, which is shown below:

  1 MKRPWSAALA ALIALGTGAS PAWAAAHPSP KVPAGAAGRV RAADVVSTPI  51SMEIQVIHDA LTVPELAAVQ AAAQAASNLS TSQWLQWLYP NATPTTSAQS 101 QAAQAVANLFNLATYGAVST RGSNAAQILQ TLQSISPLLS PRAVGLFYQS 151 FLTEIGQSSK AILARQASSSIVGNALAQAA SLSPTISAYL RQNGLSPSDL 201 ARTWSSFETQ VDPQGAAQTA LATRICTNALGFGAPTASAT ITVNTAARLR 251 TVPATAFGLN AAVWDSGLNS QTVISEVQAL HPALIRWPGGSISDVYNWET 301 NTRNDGGYVN PNDTFDNFMQ FVNAVGASPI ITVNYGTGTP QLAADWVKYA351 DVTHHDNVLY WEIGNEIYGN GYYNGNGWEA DDHAVPNQPQ KGNPGLSPQA 401YAQNALQFIQ AMRAVDPNIK IGAVLTMPYN WPWGATVNGN DDWNTVVLKA 451 LGPYIDFVDVHWYPETPGQE TDAGLLADTD QIPAMVAELK REINAYAGSN 501 AKNIQIFVTE TNSVSYNPGQQSTNLPEALF LADDLAGFVQ AGAANVDWWD 551 LLNGAEDNYT SPSLYGQNLF GDYGLLSSGQATPKGVQEPP QYTPLPPYYG 601 FQLVSDFARP GDTLLGSASS QSDIDVHAVR EPNGDIALMLVNRSPSTIYS 651 ADLNVLGVGP YAITKALVYG EGSSAVSPAL TLPTAHSVKL MPYSGVDLVL701 HPLIPAPHAA ASVTDTLALS SPTVTAGGSE TVTASFSSDR PVRDATVELE 751LYDSTGDLVA NHEMTGVDIA PGQPVSESWT FAAPAANGTY TVEAFAFDPA 801 TGATYDADTTGATITVNQPP AAKYGDIVTK NTVITVNGTT YTVPAPDASG 851 HYPSGTNISI APGDTVTIQTTFANVSSTDA LQNGLIDMEV DGQNGAIFQK 901 YWPSTTLLPG QTETVTATWQ VPSSVSAGTYPLNFQAFDTS NWTGNCYFTN 951 GGVVNFVVN

In embodiments of the invention, SEQ ID NO:307 may be glycosylated. Infurther embodiments, SEQ ID NO:307 may be glycosylated at least atpositions 174, 193, 297, 393, and 404.

With respect to the present invention, the new enzyme that was isolatedfrom an extremophilic microbe has an N-terminal sequence comprising SEQID NO:326 as shown below:

DVVSTPISMEIQV.

It will be noted, that this N-terminal sequence of the present enzymealigns/corresponds to positions 44-56 of SEQ ID NO:307.

In the present invention, an enzyme as contemplated by the presentinvention and that is isolated from an extremophilic microbe comprisesthat which is seen in SEQ ID NO:327, which is provided below:

QASSSIVGNALAQAASLSPTISAYLRQNGLSPSDLARTWSSYYCTQFDDPQGAAQTALATRICNDQALGGGAPTASATITVNTAAR.

As should be understood, this SEQ ID NO:327 aligns/corresponds topositions 166-248 of the celB sequence as seen in SEQ ID NO:307. Itshould be noted, that SEQ ID NO:327 includes changed amino acids atpositions 207, 208, 212, 229, and 231; and added amino acids atpositions 209, 213, and 230, respectively.

In the present invention, the enzyme of the present invention may befurther characterized, and is best understood by a study of SEQ IDNO:328 below:

GLNAAVWDSGLNSQTVISEVQALHPALIRWPGGSISDMDYNWETNTR

As should be understood SEQ ID NO:328, aligns/corresponds to positions258-304 of SEQ ID NO:307. It should be noted that SEQ ID NO:328 has achanged amino acid at position number 295, and an additional amino acidat position 296.

In the present invention, the enzyme as contemplated by the presentinvention further comprises the SEQ ID NO:329 as seen below:

EADDHAVPNQPQKGNPGLSPQAYAQNALQFMQSPVVYYR.

SEQ ID NO:329 aligns/corresponds to positions 379-415 of SEQ ID NO:307.It should be understood that with respect to the earlier SEQ ID NO:307,the present SEQ ID NO:329 has changes in amino acids at positions 409,411, and 413, respectively. Still further, additional amino acids arelocated at positions 412, 414, and 415, respectively.

In the prior art reference noted above to Eckert and Schneider, it isobserved that work had been conducted on the Alicyclobacillusacidocaldarius (ATCC 27009) for purposes of determining the presence ofextracellular thermoacidophilic enzymes with polysaccharide-degradingactivities. The authors noted that the organism was found to utilize avariety of polysaccharides including xylan as a sole source of carbonand energy. However, the authors failed to detect xylanase activity inthe culture supernatant. The authors assumed a cell-associated enzymeand succeeded in extracting cellulose degrading activity with associatedxylan degrading activity from the intact cells with TRITON® X-100. Theauthors observed that the cellulose degrading activity and itsassociated xylanase activity remained cell bound even after the culturereached the stationary phase of growth. In contrast, the enzyme isolatedfrom the extremophilic microbe of the present invention that displaysoptimum enzymatic activities at temperatures equal to or greater than80° C. and at a pH of less than 2, is considered to be water-soluble,and further has been isolated from cell supernatant.

The present invention is also directed to a method for the preparationof a hemicellulase that includes the steps of providing a source ofAlicyclobacillus acidocaldarius (ATCC 27009); cultivating theAlicyclobacillus acidocaldarius (ATCC 27009) in a microbial nutrientmedium having a supernatant; separating the cells of theAlicyclobacillus acidocaldarius from the nutrient medium supernatant;and recovering and purifying the hemicellulase derived from theAlicyclobacillus acidocaldarius (ATCC 27009) from the nutrient mediumsupernatant. The methodology, as described, produces a hemicellulasethat is water-soluble and displays significant enzymatic activity at apH of less than about 2, and at temperatures greater than about 80° C.In the methodology as described, the hemicellulase comprises thesequence as depicted in SEQ ID NOS:326, 327, 328, and/or 329. Stillfurther, and in the methodology as described, the nutrient medium thatis utilized further includes about 1 gram per liter of Xylan, about 10mM NH₄Cl, about 5.2 mM K₂HPO₄, about 0.8 mM MgSO₄₋₇H₂O, about 1.74 mMNa₂SO₄, about 25 mg per liter MgCl₂, about 6.6 mg per liter of CaCl₂,about 2.0 mg per liter MnSO₄, about 0.5 mg per liter ZnSO₄, about 0.5 mgper liter of boric acid, about 5 mg per liter of FeCl₃, about 0.15 mgper liter of CuSO₄, about 0.025 mg per liter of NaMoO₄, about 0.05 mgper liter of CoNO₃, about 0.02 mg per liter of NiCl₂, about 0.08 mg perliter of pyridoxine hydrochloride, about 0.01 mg per liter of folicacid, about 0.1 mg per liter of thiamine hydrochloride, about 0.04 mgper liter of riboflavin, about 0.08 mg per liter of nicotinamide, about0.08 mg per liter of p-aminobenzoate, about 0.01 mg per liter of biotin,about 0.0004 mg per liter cyanocobalamin, about 0.08 mg per literD-pantothenic acid-Ca, about 0.02 mg per liter of myo-inositol, about0.05 mg per liter of choline bromide, about 0.02 mg per liter ofmonosodium orotic acid, and about 0.1 mg per liter spermidine, whereinthe resulting nutrient medium is adjusted to a pH of about 3.5. Asdiscussed earlier in the application, the present enzyme may be used invarious processes. Therefore, the methodology, as described above,includes the step of supplying the recovered and purified hemicellulaseto a simultaneous saccharification and fermentation process tofacilitate the conversion of a biomass polysaccharide into an endproduct. One process for using the enzyme as noted above includes a stepof pretreating a biomass slurry with the recovered and purifiedhemicellulase or with a crude enzyme preparation prepared from theorganism containing a majority of the protein comprised of thehemicellulase to degrade an oligomer and/or polysaccharide that ispresent in the biomass slurry to produce an end product.

Other possible methods for using the enzyme as described above may beemployed. For example, the enzyme that has been isolated from theextremophilic microbe may be used in a method for hydrolyzing apolysaccharide, which includes the step of providing a water-solublehemicellulase derived from an extremophilic microbe; and conductinghydrolysis of a polysaccharide with the water-soluble hemicellulase at apH of less than about 2. As was discussed, earlier, the water-solublehemicellulase has an optimal enzymatic activity at a temperature ofabout 80° C.

OPERATION

The operation of the described embodiment of the present invention isbelieved to be readily apparent and is briefly summarized at this point.

As described, an enzyme isolated from extremophilic microbe thatdisplays optimum enzymatic activity at a temperature of about 80° C. anda pH of less than about 2 is best understood by a study of SEQ IDNOS:327-329, respectively. The enzyme that has been isolated is usefulin a method for treating a biomass, which includes the steps ofproviding a source of a biomass having a biomass sugar; pretreating thebiomass with a water-soluble hemicellulase derived from Alicyclobacillusacidocaldarius (ATCC 27009) to produce an end product. In the presentmethodology, the biomass sugar comprises a polysaccharide, and thehemicellulase hydrolyzes the polysaccharide. As discussed above, thehemicellulase displays enzymatic activity at a pH of less than about 2,and at temperature of greater than about 80° C.

The hemicellulase, as contemplated by the present invention, has amolecular weight of about 120 kDa. In the methodology as describedabove, the methodology includes additional steps of pretreating thebiomass in the presence or absence of the hemicellulase; providing asequential hydrolysis and fermentation process to convert the biomasssugar into the end product; and supplying the hemicellulase to thesequential hydrolysis and fermentation process to facilitate theconversion of the biomass sugar into the end product. After the step ofpretreating the biomass as discussed above, which can be performed atreduced severity in the presence or absence of the hemicellulase, themethod includes a further step of providing a simultaneoussaccharification and fermentation process to convert the biomass sugarinto the end product; and supplying the hemicellulase to thesimultaneous saccharification and fermentation process to facilitate theconversion of the biomass sugar into the end product.

Therefore, it will be seen that the present enzyme and methodology, asdescribed above, avoids many of the shortcomings attendant with theprior art enzymes and practices employed heretofore, and furtherprovides a convenient means for producing various desirable endproducts, while simultaneously reducing the severity of pretreatmentsteps that had the propensity for generating various deleterious wasteproducts, as well as for increasing the cost of the overall processthrough the requirement of high temperatures, pressures and quantitiesof acid to attain the high pretreatment severity.

Embodiments of the invention include methods of post-translationallymodifying proteins. In some embodiments, the post-translationalmodification may occur using isolated or partially purifiedglycosyltransferases and/or post-translational modification proteins,extracts of cells comprising glycosyltransferases and/orpost-translational modification proteins, and/or in cells comprising oneor more glycosyltransferases and/or post-translational modificationproteins. Glycosyltransferases and/or post-translational modificationproteins may be, without limitation, of the following classes: UDPbeta-glucosephosphotransferases, Dolichol-phosphatemannosyltransferases, and Glycosyltransferases. In some embodiments, theglycosyltransferases and/or post-translational modification proteins maybe those of a thermoacidophilic organism. Examples of thermoacidophiles,include, but are not limited to, Alicyclobacillus acidocaldarius, andthose organisms belonging to the genera Acidianus, Alicyclobacillus,Desulfurolobus, Stygiolobus, Sulfolobus, Sulfurisphaera, Sulfurococcus,Thermoplasma, and Picrophilus. Examples of glycosyltransferases and/orpost-translational modification proteins include, but are not limitedto, those provided by SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137,154, 171, 188, 205, 222, 239, 256, 273, and 290, as well as thoseavailable from the NCBI at accession numbers XP_(—)002490630.1,Q54IL5.1, ABN66322.2, XP_(—)002493240.1, XP_(—)002491463.1,XP_(—)002491326.1, CAY71061.1, NP_(—)344219.1, NP_(—)343051.1,ACR41289.1, ACP37471.1, NP_(—)111396.1, NP_(—)111382.1, NP_(—)394039.1,NP_(—)394183.1, YP 023099.1, P_(—)023093.1, AAT43750.1, and AAT42890.1.Embodiments of the invention also include proteins glycosylatedaccording to methods of the invention. Examples of such proteinsinclude, but are not limited to, glycosylated forms of the proteins ofSEQ ID NOS:307, 331, 333, 335, 337, and 338.

Embodiments of the invention include methods of altering the physicaland/or kinetic properties of an endoglucanase and/or xylanase from athermoacidophile. In some embodiments, the ratio of cellulase activityto xylanase activity of an endoglucanase and/or xylanase from athermoacidophile is altered by post-translational modification of theendoglucanase and/or xylanase. In some embodiments, thepost-translational modification may be glycosylation. In furtherembodiments, the solubility of an endoglucanase and/or xylanase from athermoacidophile is altered by post-translational modification of theendoglucanase and/or xylanase. In some embodiments, the endoglucanaseand/or xylanase is an endoglucanase and/or xylanase of Alicyclobacillusacidocaldarius. In further embodiments, the endoglucanase and/orxylanase is celB (SEQ ID NO:307).

Embodiments of the invention include modification of the ratio ofcellulase activity to xylanase activity of SEQ ID NO:307 throughglycosylation of SEQ ID NO:307. Glycosylation of SEQ ID NO:307 may beperformed, by way of non-limiting example, by expression of a sequenceencoding SEQ ID NO:307 in an organism capable of glycosylating SEQ IDNO:307, by exposure of SEQ ID NO:307 to an enzyme having glycosylatingactivity or a cell producing an enzyme having glycosylating activity; orby chemical methods known in the art.

In further embodiments of the invention, the ratio of cellulase activityto xylanase activity of an endoglucanase and/or xylanase may be alteredbased on the level and location of post-translational modification. Insome embodiments, the level and location of post-translationalmodification may be manipulated, by way of non-limiting examples,through the use of different enzymes having glycosylating activity,different cells capable of glycosylating a protein, different chemicalmethods of glycosylation, and/or by varying the amount of glycosylatingactivity or time the endoglucanase and/or xylanase is exposed to. Insome embodiments, post-translational modification of the endoglucanaseand/or xylanase results in increased xylanase activity at an acidic pH.In some embodiments, the modified form of the endoglucanase and/orxylanase has increased xylanase activity compared to an un-modified formof the endoglucanase and/or xylanase at, by way of non-limiting example,pH of less than about 5, pH of about 5, pH of about 3.5, and pH of about2.

In some embodiments, post-translational modification of theendoglucanase and/or xylanase results in greater solubility at an acidicpH. In some embodiments, the modified form of the endoglucanase and/orxylanase is more soluble that the un-modified form of the endoglucanaseand/or xylanase at, by way of non-limiting example, pH of less thanabout 5, pH of about 5, pH of about 3.5, and pH of about 2.

Embodiments of the invention include genes and associated proteinsrelated to the glycosylation and/or post-translational modification ofproteins of the thermoacidophile Alicyclobacillus acidocaldarius. Codingsequences for genes related to these processes were determined fromsequence information generated from sequencing the genome ofAlicyclobacillus acidocaldarius. These genes and proteins may representtargets for metabolic engineering of Alicyclobacillus acidocaldarius orother organisms. Non-limiting examples of nucleotide sequences foundwithin the genome of Alicyclobacillus acidocaldarius, and amino acidscoded thereby, associated with glycosylation and/or post-translationalmodification of proteins are listed in Table 1. Glycosyltransferasesand/or post-translational modification proteins may be, withoutlimitation, of the following classes: UDPbeta-glucosephosphotransferases, Dolichol-phosphatemannosyltransferases, and Glycosyltransferases; and others.

Embodiments of the invention relate in part to the gene sequences and/orprotein sequences comprising genes and/or proteins of Alicyclobacillusacidocaldarius. Genes and proteins included are those which play a rolein glycosylation and/or post-translational modification of proteins.Intracellular enzyme activities may be thermophilic and/or acidophilicin nature and general examples of similar genes are described in theliterature. Classes of genes, sequences, enzymes and factors include,but are not limited to, those listed in Table 1.

TABLE 1 Alicyclobacillus acidocaldarius genes and proteins related toglycosylation Reference Protein Sequence Gene Sequence FunctionRAAC00164 SEQ ID NO: 1 SEQ ID NO: 2 Glycosyltransferase RAAC00517 SEQ IDNO: 18 SEQ ID NO: 19 Glycosyltransferase RAAC00650 SEQ ID NO: 35 SEQ IDNO: 36 Glycosyltransferase RAAC00991 SEQ ID NO: 52 SEQ ID NO: 53Glycosyltransferase RAAC01110 SEQ ID NO: 69 SEQ ID N0: 70Glycosyltransferase RAAC01166 SEQ ID NO: 86 SEQ ID NO: 87 UDPbeta-glucose- phosphotransferase RAAC01167 SEQ ID NO: 103 SEQ ID NO: 104Glycosyltransferase RAAC01170 SEQ ID NO: 120 SEQ ID NO: 121Glycosyltransferase RAAC01248 SEQ ID NO: 137 SEQ ID NO: 138Glycosyltransferase RAAC01348 SEQ ID NO: 154 SEQ ID NO: 155Glycosyltransferase RAAC01377 SEQ ID NO: 171 SEQ ID NO: 172Glycosyltransferase RAAC01611 SEQ ID NO: 188 SEQ ID NO: 189Glycosyltransferase RAAC01612 SEQ ID NO: 205 SEQ ID NO: 206Glycosyltransferase RAAC01926 SEQ ID NO: 222 SEQ ID NO: 223Glycosyltransferase RAAC01998 SEQ ID NO: 239 SEQ ID NO: 240Glycosyltransferase RAAC02011 SEQ ID NO: 256 SEQ ID NO: 257Dolichol-phosphate mannosyltransferase RAAC02381 SEQ ID NO: 273 SEQ IDNO: 274 Glycosyltransferase RAAC02421 SEQ ID NO: 290 SEQ ID NO: 291Glycosyltransferase

The present invention relates to nucleotides sequences comprisingisolated and/or purified nucleotide sequences of the genome ofAlicyclobacillus acidocaldarius selected from the sequences of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 332, 334, 336, 339, and 340, or one of their fragments.

The present invention likewise relates to isolated and/or purifiednucleotide sequences, characterized in that they comprise at least oneof: a) a nucleotide sequence of at least one of the sequences of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 332, 334, 336, 339, and 340 or one of their fragments; b)a nucleotide sequence homologous to a nucleotide sequence such asdefined in a); c) a nucleotide sequence complementary to a nucleotidesequence such as defined in a) or b), and a nucleotide sequence of theircorresponding RNA; d) a nucleotide sequence capable of hybridizing understringent conditions with a sequence such as defined in a), b) or c); e)a nucleotide sequence comprising a sequence such as defined in a), b),c) or d); and f) a nucleotide sequence modified by a nucleotide sequencesuch as defined in a), b), c), d) or e).

Nucleotide, polynucleotide, or nucleic acid sequence will be understoodaccording to the present invention as meaning both a double-stranded ora single-stranded nucleic acid in the monomeric and dimeric (so-called“in tandem”) forms and the transcription products of the nucleic acids.

Aspects of the invention relate nucleotide sequences which it has beenpossible to isolate, purify or partially purify, starting fromseparation methods such as, for example, ion-exchange chromatography, byexclusion based on molecular size, or by affinity, or alternatively,fractionation techniques based on solubility in different solvents, orstarting from methods of genetic engineering such as amplification,cloning, and subcloning, it being possible for the sequences of theinvention to be carried by vectors.

An isolated and/or purified nucleotide sequence fragment according tothe invention will be understood as designating any nucleotide fragmentof the genome of Alicyclobacillus acidocaldarius, and may include, byway of non-limiting examples, length of at least 8, 12, 20 25, 50, 75,100, 200, 300, 400, 500, 1000, or more, consecutive nucleotides of thesequence from which it originates.

Specific fragment of an isolated and/or purified nucleotide sequenceaccording to the invention will be understood as designating anynucleotide fragment of the genome of Alicyclobacillus acidocaldarius,having, after alignment and comparison with the corresponding fragmentsof genomic sequences of Alicyclobacillus acidocaldarius, at least onenucleotide or base of different nature.

An homologous isolated and/or purified nucleotide sequence in the senseof the present invention is understood as meaning an isolated and/orpurified nucleotide sequence having at least a percentage identity withthe bases of a nucleotide sequence according to the invention of atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7%, thispercentage being purely statistical and it being possible to distributethe differences between the two nucleotide sequences at random and overthe whole of their length.

Specific homologous nucleotide sequence in the sense of the presentinvention is understood as meaning a homologous nucleotide sequencehaving at least one nucleotide sequence of a specific fragment, such asdefined above. The “specific” homologous sequences can comprise, forexample, the sequences corresponding to the genomic sequence or to thesequences of its fragments representative of variants of the genome ofAlicyclobacillus acidocaldarius. These specific homologous sequences canthus correspond to variations linked to mutations within strains ofAlicyclobacillus acidocaldarius, and especially correspond totruncations, substitutions, deletions and/or additions of at least onenucleotide. The homologous sequences can likewise correspond tovariations linked to the degeneracy of the genetic code.

The term “degree or percentage of sequence homology” refers to “degreeor percentage of sequence identity between two sequences after optimalalignment” as defined in the present application.

Two amino-acids or nucleotidic sequences are said to be “identical” ifthe sequence of amino-acids or nucleotidic residues, in the twosequences is the same when aligned for maximum correspondence, asdescribed below. Sequence comparisons between two (or more) peptides orpolynucleotides are typically performed by comparing sequences of twooptimally aligned sequences over a segment or “comparison window” toidentify and compare local regions of sequence similarity. Optimalalignment of sequences for comparison may be conducted by the localhomology algorithm of Smith and Waterman, Ad. App. Math. 2:482 (1981),by the homology alignment algorithm of Neddleman and Wunsch, J. Mol.Biol. 48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), by computerizedimplementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group (GCG),575 Science Dr., Madison, Wis.), or by visual inspection.

“Percentage of sequence identity” (or degree of identity) is determinedby comparing two optimally aligned sequences over a comparison window,where the portion of the peptide or polynucleotide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalamino-acid residue or nucleic acid base occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity.

The definition of sequence identity given above is the definition thatwould be used by one of skill in the art. The definition by itself doesnot need the help of any algorithm, the algorithms being helpful only toachieve the optimal alignments of sequences, rather than the calculationof sequence identity.

From the definition given above, it follows that there is a well definedand only one value for the sequence identity between two comparedsequences which value corresponds to the value obtained for the best oroptimal alignment.

In the BLAST N or BLAST P “BLAST 2 sequence,” software which isavailable at the web site worldwideweb.ncbi.nlm.nih.gov/gorf/b12.html,and habitually used by the inventors and in general by the skilledperson for comparing and determining the identity between two sequences,gap cost which depends on the sequence length to be compared is directlyselected by the software (i.e., 11.2 for substitution matrix BLOSUM-62for length>85).

Complementary nucleotide sequence of a sequence of the invention isunderstood as meaning any DNA whose nucleotides are complementary tothose of the sequence of the invention, and whose orientation isreversed (antisense sequence).

Hybridization under conditions of stringency with a nucleotide sequenceaccording to the invention is understood as meaning hybridization underconditions of temperature and ionic strength chosen in such a way thatthey allow the maintenance of the hybridization between two fragments ofcomplementary DNA.

By way of illustration, conditions of great stringency of thehybridization step with the aim of defining the nucleotide fragmentsdescribed above are advantageously the following.

The hybridization is carried out at a preferential temperature of 65° C.in the presence of SSC buffer, 1×SSC corresponding to 0.15 M NaCl and0.05 M Na citrate. The washing steps, for example, can be the following:2×SSC, at ambient temperature followed by two washes with 2×SSC, 0.5%SDS at 65° C.; 2×0.5×SSC, 0.5% SDS; at 65° C. for 10 minutes each.

The conditions of intermediate stringency, using, for example, atemperature of 42° C. in the presence of a 2×SSC buffer, or of lessstringency, for example a temperature of 37° C. in the presence of a2×SSC buffer, respectively, require a globally less significantcomplementarity for the hybridization between the two sequences.

The stringent hybridization conditions described above for apolynucleotide with a size of approximately 350 bases will be adapted bya person skilled in the art for oligonucleotides of greater or smallersize, according to the teachings of Sambrook et al., 1989.

Among the isolated and/or purified nucleotide sequences according to theinvention, are those that can be used as a primer or probe in methodsallowing the homologous sequences according to the invention to beobtained. These methods, such as the polymerase chain reaction (PCR),nucleic acid cloning, and sequencing, are well known to a person skilledin the art.

Among the isolated and/or purified nucleotide sequences according to theinvention, those are again preferred which can be used as a primer orprobe in methods allowing the presence of SEQ ID NOS:2, 19, 36, 53, 70,87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332,334, 336, 339, and 340, one of their fragments, or one of their variantssuch as defined below to be diagnosed.

The nucleotide sequence fragments according to the invention can beobtained, for example, by specific amplification, such as PCR, or afterdigestion with appropriate restriction enzymes of nucleotide sequencesaccording to the invention, these methods in particular being describedin the work of Sambrook et al., 1989. Such representative fragments canlikewise be obtained by chemical synthesis according to methods wellknown to persons of ordinary skill in the art.

Modified nucleotide sequence will be understood as meaning anynucleotide sequence obtained by mutagenesis according to techniques wellknown to the person skilled in the art, and containing modificationswith respect to the normal sequences according to the invention, forexample mutations in the regulatory and/or promoter sequences ofpolypeptide expression, especially leading to a modification of the rateof expression of the polypeptide or to a modulation of the replicativecycle.

Modified nucleotide sequence will likewise be understood as meaning anynucleotide sequence coding for a modified polypeptide such as definedbelow.

The present invention relates to nucleotide sequence comprising isolatedand/or purified nucleotide sequences of Alicyclobacillus acidocaldarius,characterized in that they are selected from the sequences SEQ ID NOS:2,19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257,274, 291, 332, 334, 336, 339, and 340, or one of their fragments.

Embodiments of the invention likewise relate to isolated and/or purifiednucleotide sequences characterized in that they comprise a nucleotidesequence selected from: a) at least one of a nucleotide sequence of SEQID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223,240, 257, 274, 291, 332, 334, 336, 339, and 340, or one of theirfragments; b) a nucleotide sequence of a specific fragment of a sequencesuch as defined in a); c) a homologous nucleotide sequence having atleast 80% identity with a sequence such as defined in a) or b); d) acomplementary nucleotide sequence or sequence of RNA corresponding to asequence such as defined in a), b) or c); and e) a nucleotide sequencemodified by a sequence such as defined in a), b), c) or d).

Among the isolated and/or purified nucleotide sequences according to theinvention are the nucleotide sequences of SEQ ID NOS:2, 19, 36, 53, 70,87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332,334, 336, 339, and 340, or fragments thereof and any isolated and/orpurified nucleotide sequences which have a homology of at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7% identity with the atleast one of the sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104,121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336,339, and 340, or fragments thereof. The homologous sequences cancomprise, for example, sequences corresponding to genomic sequencesAlicyclobacillus acidocaldarius. In the same manner, these specifichomologous sequences can correspond to variations linked to mutationswithin strains of Alicyclobacillus acidocaldarius and especiallycorrespond to truncations, substitutions, deletions and/or additions ofat least one nucleotide. As will be apparent to one of ordinary skill inthe art, such homologues are easily created and identified usingstandard techniques and publicly available computer programs such asBLAST. As such, each homologue referenced above should be considered asset forth herein and fully described.

Embodiments of the invention comprise the isolated and/or purifiedpolypeptides coded for by a nucleotide sequence according to theinvention, or fragments thereof, whose sequence is represented by afragment. Amino acid sequences corresponding to the isolated and/orpurified polypeptides may be coded by one of the three possible readingframes of at least one of the sequences of SEQ ID NOS:2, 19, 36, 53, 70,87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332,334, 336, 339, and 340.

Embodiments of the invention likewise relate to the isolated and/orpurified polypeptides, characterized in that they comprise a polypeptideselected from at least one of the amino acid sequences of SEQ ID NOS:1,18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256,273, 290, 307, 331, 333, 335, 337, and 338, or one of their fragments.

Among the isolated and/or purified polypeptides, according toembodiments of the invention, are the isolated and/or purifiedpolypeptides of amino acid sequence SEQ ID NOS:1, 18, 35, 52, 69, 86,103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 331,333, 335, 337, and 338, or fragments thereof or any other isolatedand/or purified polypeptides which have a homology of at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7% identity with at least one ofthe sequences of SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154,171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333, 335, 337, and338, or fragments thereof. As will be apparent to one of ordinary skillin the art, such homologues are easily created and identified usingstandard techniques and publicly available computer programs such asBLAST. As such, each homologue referenced above should be considered asset forth herein and fully described.

Embodiments of the invention also relate to the polypeptides,characterized in that they comprise a polypeptide selected from: a) aspecific fragment of at least 5 amino acids of a polypeptide of an aminoacid sequence according to the invention; b) a polypeptide homologous toa polypeptide such as defined in a); c) a specific biologically activefragment of a polypeptide such as defined in a) or b); and d) apolypeptide modified by a polypeptide such as defined in a), b) or c).

In the present description, the terms polypeptide, peptide and proteinare interchangeable.

In embodiments of the invention, the isolated and/or purifiedpolypeptides according to the invention may be glycosylated, pegylated,and/or otherwise post-translationally modified. In further embodiments,glycosylation, pegylation, and/or other post-translational modificationsmay occur in vivo or in vitro and/or may be performed using chemicaltechniques. In additional embodiments, any glycosylation, pegylationand/or other post-translational modifications may be N-linked orO-linked.

In embodiments of the invention, any one of the isolated and/or purifiedpolypeptides according to the invention may be enzymatically orfunctionally active at temperatures at or above about 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/ormay be enzymatically or functionally active at a pH at, below, and/orabove 8, 7, 6, 5, 4, 3, 2, 1, and/or 0. In further embodiments of theinvention, glycosylation, pegylation, and/or other post-translationalmodification may be required for the isolated and/or purifiedpolypeptides according to the invention to be enzymatically orfunctionally active at a pH at or below 8, 7, 6, 5, 4, 3, 2, 1, and/or 0or at temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, and/or 95 degrees Celsius.

Aspects of the invention relate to polypeptides that are isolated orobtained by purification from natural sources, or else obtained bygenetic recombination, or alternatively by chemical synthesis and thatthey may thus contain unnatural amino acids, as will be described below.

A “polypeptide fragment” according to the embodiments of the inventionis understood as designating a polypeptide containing at least 5consecutive amino acids, preferably 10 consecutive amino acids or 15consecutive amino acids.

In the present invention, a “specific polypeptide fragment” isunderstood as designating the consecutive polypeptide fragment coded forby a specific fragment of a nucleotide sequence according to theinvention.

“Homologous polypeptide” will be understood as designating thepolypeptides having, with respect to the natural polypeptide, certainmodifications such as, in particular, a deletion, addition, orsubstitution of at least one amino acid, a truncation, a prolongation, achimeric fusion, and/or a mutation. Among the homologous polypeptides,those are preferred whose amino acid sequence has at least 80% or 90%,homology with the sequences of amino acids of polypeptides according tothe invention.

“Specific homologous polypeptide” will be understood as designating thehomologous polypeptides such as defined above and having a specificfragment of a polypeptide according to the invention.

In the case of a substitution, one or more consecutive or nonconsecutiveamino acids are replaced by “equivalent” amino acids. The expression“equivalent” amino acid is directed here at designating any amino acidcapable of being substituted by one of the amino acids of the basestructure without, however, essentially modifying the biologicalactivities of the corresponding peptides and such that they will bedefined by the following. As will be apparent to one of ordinary skillin the art, such substitutions are easily created and identified usingstandard molecular biology techniques and publicly available computerprograms such as BLAST. As such, each substitution referenced aboveshould be considered as set forth herein and fully described. Theseequivalent amino acids may be determined either by depending on theirstructural homology with the amino acids which they substitute, or onresults of comparative tests of biological activity between thedifferent polypeptides, which are capable of being carried out.

By way of nonlimiting example, the possibilities of substitutionscapable of being carried out without resulting in an extensivemodification of the biological activity of the corresponding modifiedpolypeptides will be mentioned, the replacement, for example, of leucineby valine or isoleucine, of aspartic acid by glutamic acid, of glutamineby asparagine, of arginine by lysine etc., the reverse substitutionsnaturally being envisageable under the same conditions.

In a further embodiment, substitutions are limited to substitutions inamino acids not conserved among other proteins which have similaridentified enzymatic activity. For example, one of ordinary skill in theart may align proteins of the same function in similar organisms anddetermine which amino acids are generally conserved among proteins ofthat function. One example of a program that may be used to generatesuch alignments is available on the Internet atworldwideweb.charite.de/bioinf/strap/in conjunction with the databasesprovided by the NCBI.

Thus, according to one embodiment of the invention, substitutions ormutations may be made at positions that are generally conserved amongproteins of that function. In a further embodiment, nucleic acidsequences may be mutated or substituted such that the amino acid theycode for is unchanged (degenerate substitutions and/mutations) and/ormutated or substituted such that any resulting amino acid substitutionsor mutations are made at positions that are generally conserved amongproteins of that function.

The specific homologous polypeptides likewise correspond to polypeptidescoded for by the specific homologous nucleotide sequences such asdefined above and, thus, comprise in the present definition thepolypeptides that are mutated or correspond to variants which can existin Alicyclobacillus acidocaldarius, and which especially correspond totruncations, substitutions, deletions, and/or additions of at least oneamino acid residue.

“Specific biologically active fragment of a polypeptide” according to anembodiment of the invention will be understood in particular asdesignating a specific polypeptide fragment, such as defined above,having at least one of the characteristics of polypeptides according tothe invention. In certain embodiments, the peptide is capable ofbehaving as at least one of the types of proteins outlined in Table 1.

The polypeptide fragments according to embodiments of the invention, cancorrespond to isolated or purified fragments naturally present inAlicyclobacillus acidocaldarius or correspond to fragments which can beobtained by cleavage of the polypeptide by a proteolytic enzyme, such astrypsin or chymotrypsin or collagenase, or by a chemical reagent, suchas cyanogen bromide (CNBr). Such polypeptide fragments can likewise justas easily be prepared by chemical synthesis, from hosts transformed byan expression vector, according to the invention, containing a nucleicacid allowing the expression of the fragments, placed under the controlof appropriate regulation and/or expression elements.

“Modified polypeptide” of a polypeptide according to an embodiment ofthe invention is understood as designating a polypeptide obtained bygenetic recombination or by chemical synthesis as will be describedbelow, having at least one modification with respect to the normalsequence. These modifications may or may not be able to bear on aminoacids at the origin of specificity, and/or of activity, or at the originof the structural conformation, localization, and of the capacity ofmembrane insertion of the polypeptide according to the invention. Itwill thus be possible to create polypeptides of equivalent, increased,or decreased activity, and of equivalent, narrower, or widerspecificity. Among the modified polypeptides, it is necessary to mentionthe polypeptides in which up to 5 or more amino acids can be modified,truncated at the N- or C-terminal end, or even deleted or added.

The methods allowing the modulations on eukaryotic or prokaryotic cellsto be demonstrated are well known to the person of ordinary skill in theart. It is likewise well understood that it will be possible to use thenucleotide sequences coding for the modified polypeptides for themodulations, for example, through vectors according to the invention anddescribed below.

The preceding modified polypeptides can be obtained by usingcombinatorial chemistry, in which it is possible to systematically varyparts of the polypeptide before testing them on models, cell cultures ormicroorganisms, for example, to select the compounds that are mostactive or have the properties sought.

Chemical synthesis likewise has the advantage of being able to useunnatural amino acids, or nonpeptide bonds.

Thus, in order to improve the duration of life of the polypeptidesaccording to the invention, it may be of interest to use unnatural aminoacids, for example in D form, or else amino acid analogs, especiallysulfur-containing forms, for example.

Finally, it will be possible to integrate the structure of thepolypeptides according to the invention, its specific or modifiedhomologous forms, into chemical structures of polypeptide type orothers. Thus, it may be of interest to provide at the N- and C-terminalends molecules not recognized by proteases.

The nucleotide sequences coding for a polypeptide according to theinvention are likewise part of the invention.

The invention likewise relates to nucleotide sequences utilizable as aprimer or probe, characterized in that the sequences are selected fromthe nucleotide sequences according to the invention.

It is well understood that the present invention, in variousembodiments, likewise relates to specific polypeptides ofAlicyclobacillus acidocaldarius, coded for by nucleotide sequences,capable of being obtained by purification from natural polypeptides, bygenetic recombination or by chemical synthesis by procedures well knownto a person skilled in the art and such as described in particularbelow. In the same manner, the labeled or unlabeled mono- or polyclonalantibodies directed against the specific polypeptides coded for by thenucleotide sequences are also encompassed by the invention.

Embodiments of the invention additionally relate to the use of anucleotide sequence according to the invention as a primer or probe forthe detection and/or the amplification of nucleic acid sequences.

The nucleotide sequences according to embodiments of the invention canthus be used to amplify nucleotide sequences, especially by the PCRtechnique (Polymerase Chain Reaction) (Erlich, 1989; Innis et al., 1990;Rolfs et al., 1991; and White et al., 1997).

These oligodeoxyribonucleotide or oligoribonucleotide primersadvantageously have a length of at least 8 nucleotides, preferably of atleast 12 nucleotides, and even more preferentially at least 20nucleotides.

Other amplification techniques of the target nucleic acid can beadvantageously employed as alternatives to PCR.

The nucleotide sequences of the invention, in particular the primersaccording to the invention, can likewise be employed in other proceduresof amplification of a target nucleic acid, such as: the TAS technique(Transcription-based Amplification System), described by Kwoh et al. in1989; the 3SR technique (Self-Sustained Sequence Replication), describedby Guatelli et al. in 1990; the NASBA technique (Nucleic Acid SequenceBased Amplification), described by Kievitis et al. in 1991; the SDAtechnique (Strand Displacement Amplification) (Walker et al., 1992); andthe TMA technique (Transcription Mediated Amplification).

The polynucleotides of the invention can also be employed in techniquesof amplification or of modification of the nucleic acid serving as aprobe, such as: the LCR technique (Ligase Chain Reaction), described byLandegren et al. in 1988 and improved by Barany et al. in 1991, whichemploys a thermostable ligase; the RCR technique (Repair ChainReaction), described by Segev in 1992; the CPR technique (Cycling ProbeReaction), described by Duck et al. in 1990; the amplification techniquewith Q-beta replicase, described by Miele et al. in 1983 and especiallyimproved by Chu et al. in 1986, Lizardi et al. in 1988, then by Burg etal., as well as by Stone et al. in 1996.

In the case where the target polynucleotide to be detected is possiblyan RNA, for example an mRNA, it will be possible to use, prior to theemployment of an amplification reaction with the aid of at least oneprimer according to the invention or to the employment of a detectionprocedure with the aid of at least one probe of the invention, an enzymeof reverse transcriptase type in order to obtain a cDNA from the RNAcontained in the biological sample. The cDNA obtained will thus serve asa target for the primer(s) or the probe(s) employed in the amplificationor detection procedure according to the invention.

The detection probe will be chosen in such a manner that it hybridizeswith the target sequence or the amplicon generated from the targetsequence. By way of sequence, such a probe will advantageously have asequence of at least 12 nucleotides, in particular of at least 20nucleotides, and preferably of at least 100 nucleotides.

Embodiments of the invention also comprise the nucleotide sequencesutilizable as a probe or primer according to the invention,characterized in that they are labeled with a radioactive compound orwith a nonradioactive compound.

The unlabeled nucleotide sequences can be used directly as probes orprimers, although the sequences are generally labeled with a radioactiveisotope (32P, 35S, 3H, 125I) or with a nonradioactive molecule (biotin,acetylaminofluorene, digoxigenin, 5-bromodeoxyuridine, fluorescein) toobtain probes which are utilizable for numerous applications.

Examples of nonradioactive labeling of nucleotide sequences aredescribed, for example, in French Patent No. 7810975 or by Urdea et al.or by Sanchez-Pescador et al. in 1988.

In the latter case, it will also be possible to use one of the labelingmethods described in French Patent Appl. Publication Nos. FR-2422956 andFR-2518755.

The hybridization technique can be carried out in various manners(Matthews et al., 1988). The most general method consists inimmobilizing the nucleic acid extract of cells on a support (such asnitrocellulose, nylon, polystyrene) and in incubating, underwell-defined conditions, the immobilized target nucleic acid with theprobe. After hybridization, the excess of the probe is eliminated andthe hybrid molecules formed are detected by the appropriate method(measurement of the radioactivity, of the fluorescence or of theenzymatic activity linked to the probe).

The invention, in various embodiments, likewise comprises the nucleotidesequences according to the invention, characterized in that they areimmobilized on a support, covalently or noncovalently.

According to another advantageous mode of employing nucleotide sequencesaccording to the invention, the latter can be used immobilized on asupport and can thus serve to capture, by specific hybridization, thetarget nucleic acid obtained from the biological sample to be tested. Ifnecessary, the solid support is separated from the sample and thehybridization complex formed between the capture probe and the targetnucleic acid is then detected with the aid of a second probe, aso-called detection probe, labeled with an easily detectable element.

Another aspect of the present invention is a vector for the cloningand/or expression of a sequence, characterized in that it contains anucleotide sequence according to the invention.

The vectors according to the invention, characterized in that theycontain the elements allowing the integration, expression and/or thesecretion of the nucleotide sequences in a determined host cell, arelikewise part of the invention.

The vector may then contain a promoter, signals of initiation andtermination of translation, as well as appropriate regions of regulationof transcription. The vector may be able to be maintained stably in thehost cell and can optionally have particular signals specifying thesecretion of the translated protein. These different elements may bechosen as a function of the host cell used. To this end, the nucleotidesequences according to the invention may be inserted into autonomousreplication vectors within the chosen host, or integrated vectors of thechosen host.

Such vectors will be prepared according to the methods currently used bya person skilled in the art, and it will be possible to introduce theclones resulting therefrom into an appropriate host by standard methods,such as, for example, lipofection, electroporation, and thermal shock.

The vectors according to the invention are, for example, vectors ofplasmid or viral origin. One example of a vector for the expression ofpolypeptides of the invention is baculovirus.

These vectors are useful for transforming host cells in order to cloneor to express the nucleotide sequences of the invention.

The invention likewise comprises the host cells transformed by a vectoraccording to the invention.

These cells can be obtained by the introduction into host cells of anucleotide sequence inserted into a vector such as defined above, thenthe culturing of the cells under conditions allowing the replicationand/or expression of the transfected nucleotide sequence.

The host cell can be selected from prokaryotic or eukaryotic systems,such as, for example, bacterial cells (Olins and Lee, 1993), butlikewise yeast cells (Buckholz, 1993), as well as plant cells, such asArabidopsis sp., and animal cells, in particular the cultures ofmammalian cells (Edwards and Aruffo, 1993), for example, Chinese hamsterovary (CHO) cells, but likewise the cells of insects in which it ispossible to use procedures employing baculoviruses, for example, Sf9insect cells (Luckow, 1993).

Embodiments of the invention likewise relate to organisms comprising oneof the transformed cells according to the invention.

The obtainment of transgenic organisms according to the inventionexpressing one or more of the genes of Alicyclobacillus acidocaldarius,or part of the genes, may be carried out in, for example, rats, mice, orrabbits according to methods well known to a person skilled in the art,such as by viral or nonviral transfections. It will be possible toobtain the transgenic organisms expressing one or more of the genes bytransfection of multiple copies of the genes under the control of astrong promoter of ubiquitous nature, or selective for one type oftissue. It will likewise be possible to obtain the transgenic organismsby homologous recombination in embryonic cell strains, transfer of thesecell strains to embryos, selection of the affected chimeras at the levelof the reproductive lines, and growth of the chimeras.

The transformed cells as well as the transgenic organisms according tothe invention are utilizable in procedures for preparation ofrecombinant polypeptides.

It is today possible to produce recombinant polypeptides in relativelylarge quantities by genetic engineering using the cells transformed byexpression vectors according to the invention or using transgenicorganisms according to the invention.

The procedures for preparation of a polypeptide of the invention inrecombinant form, characterized in that they employ a vector and/or acell transformed by a vector according to the invention and/or atransgenic organism comprising one of the transformed cells according tothe invention are themselves comprised in the present invention.

As used herein, “transformation” and “transformed” relate to theintroduction of nucleic acids into a cell, whether prokaryotic oreukaryotic. Further, “transformation” and “transformed,” as used herein,need not relate to growth control or growth deregulation.

Among the procedures for preparation of a polypeptide of the inventionin recombinant form, the preparation procedures employing a vector,and/or a cell transformed by the vector and/or a transgenic organismcomprising one of the transformed cells, containing a nucleotidesequence according to the invention coding for a polypeptide ofAlicyclobacillus acidocaldarius.

A variant according to an embodiment of the invention may consist ofproducing a recombinant polypeptide fused to a “carrier” protein(chimeric protein). The advantage of this system is that it may allowstabilization of and/or a decrease in the proteolysis of the recombinantproduct, an increase in the solubility in the course of renaturation invitro and/or a simplification of the purification when the fusionpartner has an affinity for a specific ligand.

More particularly, an embodiment of the invention relates to a procedurefor preparation of a polypeptide of the invention comprising thefollowing steps: a) culture of transformed cells under conditionsallowing the expression of a recombinant polypeptide of nucleotidesequence according to the invention; b) if need be, recovery of therecombinant polypeptide.

When the procedure for preparation of a polypeptide of the inventionemploys a transgenic organism according to the invention, therecombinant polypeptide is then extracted from the organism.

An embodiment of the invention also relates to a polypeptide which iscapable of being obtained by a procedure of the invention such asdescribed previously.

The invention, in another embodiment, also comprises a procedure forpreparation of a synthetic polypeptide, characterized in that it uses asequence of amino acids of polypeptides according to the invention.

A further embodiment of the invention likewise relates to a syntheticpolypeptide obtained by a procedure according to the invention.

The polypeptides according to embodiments of the invention can likewisebe prepared by techniques which are conventional in the field of thesynthesis of peptides. This synthesis can be carried out in ahomogeneous solution or in a solid phase.

For example, recourse can be made to the technique of synthesis in ahomogeneous solution described by Houben-Weyl in 1974.

This method of synthesis comprises successively condensing, two by two,the successive amino acids in the order required, or in condensing aminoacids and fragments formed previously and already containing severalamino acids in the appropriate order, or alternatively several fragmentspreviously prepared in this way, it being understood that it will benecessary to protect beforehand all the reactive functions carried bythese amino acids or fragments, with the exception of amine functions ofone and carboxyls of the other or vice-versa, which must normally beinvolved in the formation of peptide bonds, especially after activationof the carboxyl function, according to the methods well known in thesynthesis of peptides.

Recourse may also be made to the technique described by Merrifield.

To make a peptide chain according to the Merrifield procedure, recourseis made to a very porous polymeric resin, on which is immobilized thefirst C-terminal amino acid of the chain. This amino acid is immobilizedon a resin through its carboxyl group and its amine function isprotected. The amino acids that are going to form the peptide chain arethus immobilized, one after the other, on the amino group, which isdeprotected beforehand each time, of the portion of the peptide chainalready formed, and which is attached to the resin. When the whole ofthe desired peptide chain has been formed, the protective groups of thedifferent amino acids forming the peptide chain are eliminated and thepeptide is detached from the resin with the aid of an acid.

The invention additionally relates to hybrid polypeptides having atleast one polypeptide according to the invention, and a sequence of apolypeptide capable of inducing an immune response in man or animals.

Advantageously, the antigenic determinant is such that it is capable ofinducing a humoral and/or cellular response.

It will be possible for such a determinant to comprise a polypeptideaccording to the invention in glycosylated, pegylated, and/or otherwisepost-translationally modified form used with a view to obtainingimmunogenic compositions capable of inducing the synthesis of antibodiesdirected against multiple epitopes.

These hybrid molecules can be formed, in part, of a polypeptide carriermolecule or of fragments thereof according to the invention, associatedwith a possibly immunogenic part, in particular an epitope of thediphtheria toxin, the tetanus toxin, a surface antigen of the hepatitisB virus (French Patent 7921811), the VP1 antigen of the poliomyelitisvirus or any other viral or bacterial toxin or antigen.

The procedures for synthesis of hybrid molecules encompass the methodsused in genetic engineering for constructing hybrid nucleotide sequencescoding for the polypeptide sequences sought. It will be possible, forexample, to refer advantageously to the technique for obtainment ofgenes coding for fusion proteins described by Minton in 1984.

The hybrid nucleotide sequences coding for a hybrid polypeptide, as wellas the hybrid polypeptides according to the invention, are socharacterized in that they are recombinant polypeptides obtained by theexpression of the hybrid nucleotide sequences, which are likewise partof the invention.

The invention likewise comprises the vectors characterized in that theycontain one of the hybrid nucleotide sequences. The host cellstransformed by the vectors, the transgenic organisms comprising one ofthe transformed cells as well as the procedures for preparation ofrecombinant polypeptides using the vectors, the transformed cells and/orthe transgenic organisms are, of course, likewise part of the invention.

The polypeptides according to the invention, the antibodies according tothe invention described below and the nucleotide sequences according tothe invention can advantageously be employed in procedures for thedetection and/or identification of Alicyclobacillus acidocaldarius, in asample capable of containing them. These procedures, according to thespecificity of the polypeptides, the antibodies and the nucleotidesequences according to the invention which will be used, will inparticular be able to detect and/or to identify Alicyclobacillusacidocaldarius.

The polypeptides according to the invention can advantageously beemployed in a procedure for the detection and/or the identification ofAlicyclobacillus acidocaldarius in a sample capable of containing them,characterized in that it comprises the following steps: a) contacting ofthis sample with a polypeptide or one of its fragments according to theinvention (under conditions allowing an immunological reaction betweenthe polypeptide and the antibodies possibly present in the biologicalsample); b) demonstration of the antigen-antibody complexes possiblyformed.

Any conventional procedure can be employed for carrying out such adetection of the antigen-antibody complexes possibly formed.

By way of example, a method according to various embodiments brings intoplay immunoenzymatic processes according to the ELISA technique, byimmunofluorescence, or radioimmunological processes (RIA) or theirequivalent.

Thus, the invention likewise relates to the polypeptides according tothe invention, labeled with the aid of an adequate label, such as, ofthe enzymatic, fluorescent or radioactive type.

Such methods comprise, for example, the following acts: deposition ofdetermined quantities of a polypeptide composition according to theinvention in the wells of a microtiter plate, introduction into thewells of increasing dilutions of serum, or of a biological sample otherthan that defined previously, having to be analyzed, incubation of themicrotiter plate, introduction into the wells of the microtiter plate oflabeled antibodies directed against pig immunoglobulins, the labeling ofthese antibodies having been carried out with the aid of an enzymeselected from those which are capable of hydrolyzing a substrate bymodifying the absorption of the radiation of the latter, at least at adetermined wavelength, for example at 550 nm, detection, by comparisonwith a control test, of the quantity of hydrolyzed substrate.

The polypeptides according to embodiments of the invention enablemonoclonal or polyclonal antibodies to be prepared which arecharacterized in that they specifically recognize the polypeptidesaccording to the invention. It will advantageously be possible toprepare the monoclonal antibodies from hybridomas according to thetechnique described by Kohler and Milstein in 1975. It will be possibleto prepare the polyclonal antibodies, for example, by immunization of ananimal, in particular a mouse, with a polypeptide or a DNA, according tothe invention, associated with an adjuvant of the immune response, andthen purification of the specific antibodies contained in the serum ofthe immunized animals on an affinity column on which the polypeptidewhich has served as an antigen has previously been immobilized. Thepolyclonal antibodies according to the invention can also be prepared bypurification, on an affinity column on which a polypeptide according tothe invention has previously been immobilized, of the antibodiescontained in the serum of an animal immunologically challenged byAlicyclobacillus acidocaldarius, or a polypeptide or fragment accordingto the invention.

The invention, in various embodiments, likewise relates to mono- orpolyclonal antibodies or their fragments, or chimeric antibodies,characterized in that they are capable of specifically recognizing apolypeptide according to the invention.

It will likewise be possible for the antibodies of embodiments of theinvention to be labeled in the same manner as described previously forthe nucleic probes of the invention, such as a labeling of enzymatic,fluorescent or radioactive type.

An embodiment of the invention is additionally directed at a procedurefor the detection and/or identification of Alicyclobacillusacidocaldarius in a sample, characterized in that it comprises thefollowing steps: a) contacting of the sample with a mono- or polyclonalantibody according to the invention (under conditions allowing animmunological reaction between the antibodies and the polypeptides ofAlicyclobacillus acidocaldarius possibly present in the biologicalsample); b) demonstration of the antigen-antibody complex possiblyformed.

The present invention likewise relates to a procedure for the detectionand/or the identification of Alicyclobacillus acidocaldarius in asample, characterized in that it employs a nucleotide sequence accordingto the invention.

More particularly, the invention relates to a procedure for thedetection and/or the identification of Alicyclobacillus acidocaldariusin a sample, characterized in that it contains the following steps: a)if need be, isolation of the DNA from the sample to be analyzed; b)specific amplification of the DNA of the sample with the aid of at leastone primer, or a pair of primers, according to the invention; c)demonstration of the amplification products.

These can be detected, for example, by the technique of molecularhybridization utilizing a nucleic probe according to the invention. Thisprobe will advantageously be labeled with a nonradioactive (cold probe)or radioactive isotope.

For the purposes of the present invention, “DNA of the biologicalsample” or “DNA contained in the biological sample” will be understoodas meaning either the DNA present in the biological sample considered,or possibly the cDNA obtained after the action of an enzyme of reversetranscriptase type on the RNA present in the biological sample.

A further embodiment of the invention comprises a method, characterizedin that it comprises the following steps: a) contacting of a nucleotideprobe according to the invention with a biological sample, the DNAcontained in the biological sample having, if need be, previously beenmade accessible to hybridization under conditions allowing thehybridization of the probe with the DNA of the sample; b) demonstrationof the hybrid formed between the nucleotide probe and the DNA of thebiological sample.

The present invention also relates to a procedure according toembodiments of the invention, characterized in that it comprises thefollowing steps: a) contacting of a nucleotide probe immobilized on asupport according to the invention with a biological sample, the DNA ofthe sample having, if need be, previously been made accessible tohybridization, under conditions allowing the hybridization of the probewith the DNA of the sample; b) contacting of the hybrid formed betweenthe nucleotide probe immobilized on a support and the DNA contained inthe biological sample, if need be after elimination of the DNA of thebiological sample which has not hybridized with the probe, with anucleotide probe labeled according to the invention; c) demonstration ofthe novel hybrid formed in step b).

According to an advantageous embodiment of the procedure for detectionand/or identification defined previously, this is characterized in that,prior to step a), the DNA of the biological sample is first amplifiedwith the aid of at least one primer according to the invention.

Embodiments of methods include glycosylating or post-translationallymodifying a first polypeptide using a second polypeptide selected fromthe group consisting of a polypeptide having at least 90% sequenceidentity to SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171,188, 205, 222, 239, 256, 273, 290, 307, 331, 333, 335, 337, and 338.

Further embodiments of methods include methods of modulating proteinstability, solubility, degradation, activity profile, and/orexternalization of a first polypeptide, the methods comprisingglycosylating or post-translationally modifying the first polypeptidevia a second polypeptide selected from the group consisting of apolypeptide having at least 90% sequence identity to SEQ ID NOS:1, 18,35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273,290, 307, 331, 333, 335, 337 and 338.

Further embodiments of methods include placing a cell producing orencoding a recombinant, purified, and/or isolated nucleotide sequencecomprising a nucleotide sequence selected from the group consisting of anucleotide sequences having at least 90% sequence identity to at leastone of the sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138,155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and340, and/or a recombinant, purified, and/or isolated polypeptideselected from the group consisting of a polypeptide having at least 90%sequence identity to at least one of the sequences of SEQ ID NOS:1, 18,35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273,290, 307, 331, 333, 335, 337, and 338 in a environment comprisingtemperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, and/or 95 degrees Celsius and/or a pH at, below, and/orabove 8, 7, 6, 5, 4, 3, 2, 1, and/or 0.

The present invention provides cells that have been geneticallymanipulated to have an altered capacity to produce expressed proteins.In particular, the present invention relates to Gram-positivemicroorganisms, such as Bacillus species having enhanced expression of aprotein of interest, wherein one or more chromosomal genes have beeninactivated, and/or wherein one or more chromosomal genes have beendeleted from the Bacillus chromosome. In some further embodiments, oneor more indigenous chromosomal regions have been deleted from acorresponding wild-type Bacillus host chromosome. In furtherembodiments, the Bacillus is an Alicyclobacillus sp. or Alicyclobacillusacidocaldarius.

In additional embodiments, methods of glycosylating and/orpost-translationally modifying a polypeptide at temperatures at or aboveabout 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95degrees Celsius and/or at a pH at, below, and/or above 8, 7, 6, 5, 4, 3,2, 1, and/or 0 via a recombinant, purified, and/or isolated nucleotidesequence comprising a nucleotide sequence selected from the groupconsisting of a nucleotide sequences having at least 90% sequenceidentity to at least one of the sequences of SEQ ID NOS:2, 19, 36, 53,70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332,334, 336, 339, and 340, and/or a recombinant, purified, and/or isolatedpolypeptide selected from the group consisting of a polypeptide havingat least 90% sequence identity to at least one of the sequences of SEQID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222,239, 256, 273, 290, 307, 331, 333, 335, 337, and 338.

In embodiments of the invention, any one of the isolated and/or purifiedpolypeptides according to the invention may be enzymatically orfunctionally active at temperatures at or above about 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/ormay be enzymatically or functionally active at a pH at, below, and/orabove 8, 7, 6, 5, 4, 3, 2, 1, and/or 0. In further embodiments of theinvention, glycosylation, pegylation, and/or other post-translationalmodification may be required for the isolated and/or purifiedpolypeptides according to the invention to be enzymatically orfunctionally active at pH at or below 8, 7, 6, 5, 4, 3, 2, 1, and/or 0or at a temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius.

The invention is described in additional detail in the followingillustrative examples. Although the examples may represent only selectedembodiments of the invention, it should be understood that the followingexamples are illustrative and not limiting.

EXAMPLES Example 1 Glycosylation Using Nucleotide and Amino AcidSequences from Alicyclobacillus acidocaldarius

Provided in SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172,189, 206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340 are anucleotide sequences isolated from Alicyclobacillus acidocaldarius andcoding for the polypeptides of SEQ ID NOS:1, 18, 35, 52, 69, 86, 103,120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333,335, 337, and 338, respectively. The nucleotide sequences of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 332, 334, 336, 339, and 340 are placed into expressionvectors using techniques standard in the art. The vectors are thenprovided to cells such as bacteria cells or eukaryotic cells such as Sf9cells or CHO cells. In conjunction with the normal machinery in presentin the cells, the vectors comprising SEQ ID NOS:2, 19, 36, 53, 70, 87,104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334,336, 339, and 340 produce the polypeptides of SEQ ID NOS:1, 18, 35, 52,69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307,331, 333, 335, 337, and 338. The polypeptides of SEQ ID NOS:1, 18, 35,52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290,307, 331, 333, 335, 337, and 338 are then isolated and/or purified. Theisolated and/or purified polypeptides of SEQ ID NOS:1, 18, 35, 52, 69,86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307,331, 333, 335, 337, and 338 are then each demonstrated to have one ormore of the activities provided in Table 1 or some other activity.

The isolated and/or purified polypeptides of SEQ ID NOS:1, 18, 35, 52,69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290 aredemonstrated to have activity in glycosylating other proteins inconjunction with other proteins or cellular components.

Example 2 Modulating Protein Stability, Solubility, Degradation,Activity Profile, and/or Externalization of a First Polypeptide UsingNucleotide and Amino Acid Sequences from Alicyclobacillus acidocaldarius

The polypeptides and nucleotide sequence of Example 1 are used topost-translationally modify one or more other proteins throughglycosylation or other post-translational modification. The modifiedproteins are demonstrated to have altered protein stability, solubility,degradation, activity profile, and/or externalization in comparison tonon-modified proteins of the same or similar amino acid sequence.

Example 3 Glycosylated Proteins of Alicyclobacillus acidocaldarius

Polypeptide RAAC02676 (SEQ ID NO:307) was obtained via the followingprotocol. Alicyclobacillus acidocaldarius was cultured on wheatarabinoxylan and harvested after three days. The culture was centrifugedto remove cells and the resulting supernatant was filtered with a 0.22micron filter to remove any remaining debris. The filtered supernatantwas concentrated to approximately 1 mL by ultrafiltration through a10,000 Da molecular weight cutoff membrane. The resulting concentratedfiltered supernatant was additionally purified by trapping proteins on acation exchange column, eluting them with a salt gradient, reloadingthem on a second cation exchange column and eluting with a second saltgradient. Samples were pooled and run on a 12% SDS-PAGE gel. Individualbands were cut from the gel and subjected to in gel tryptic digestion.The peptide fragments were then eluted and separated on a C-18 column aninjected into an ion trap mass spectrometer via electrospray. Massspectra were run through MASCOT which compares the observed spectra totheoretical spectra generated from the known protein sequence. MASCOTallows the user to specify modifications that might exist on the proteinand looks for spectra consistent with these modifications. MASCOTidentified a number of peptides digested from RAAC02676 that werepotentially glycosylated as provided in Table 2 below.

As can be seen in Table 2, Queries 94, 96, 221, 332, 333, 337, and 400returned expected N-linked glycosylations on RAAC02676. All fragments inTable 2 are fragments of SEQ ID NO:307 (RAAC02676). O-linkedglycosylations are also expected.

TABLE 2 Query Observed Mr(expt) Mr(calc) Ppm Miss Score Expect RankPeptide 17 398.1406 794.2667 794.4174 −189.65 0 (24) 73 1 K.YGDIVTK.N 18398.1591 794.3037 794.4174 −143.08 0 (28) 38 2 K.YGDIVTK.N 19 398.1596794.3047 794.4174 −141.82 0 55 0.073 1 K.YGDIVTK.N 20 398.1621 794.3097794.4174 −135.53 0 (41) 1.7 1 K.YGDIVTK.N 77 569.1781 1136.34171136.5461 −179.86 0 (46) 0.41 1 R.EINAYAGSNAK.N 78 569.1796 1136.34471136.5461 −177.22 0 62 0.011 1 R.EINAYAGSNAK.N 94 579.6776 1157.34071157.5676 −196.02 0 34 6.9 1 R.QNGLSPSDLAR.T + Glyc-Asn (N) 96 579.70711157.3997 1157.5676 −145.05 0 (20) 2.5e+02 3 R.QNGLSPSDLAR.T + Glyc-Asn(N) 138 721.2386 1440.4627 1440.7394 −192.07 0 (62) 0.019 1R.EPNGDIALMLVNR.S 139 721.2411 1440.4677 1440.7394 −188.60 0 82 0.000221 R.EPNGDIALMLVNR.S 207 987.8796 1973.7447 1974.0098 −134.27 0 (130) 6.2e−09 1 R.AVGLFYQSFLTEIGQSSK.A 208 987.8796 1973.7447 1974.0098−134.27 0 (123)  2.8e−08 1 R.AVGLFYQSFLTEIGQSSK.A 209 987.8801 1973.74571974.0098 −133.76 0 (71) 0.0045 1 R.AVGLFYQSFLTEIGQSSK.A 210 987.89861973.7827 1974.0098 −115.02 0 140  5.9e−10 1 R.AVGLFYQSFLTEIGQSSK.A 216661.1822 1980.5247 1980.8966 −187.71 0 (37) 4.1 1 R.WPGGSISDVYNWETNTR.N217 991.3196 1980.6247 1980.8966 −137.23 0 (59) 0.053 1R.WPGGSISDVYNWETNTR.N 218 991.3411 1980.6677 1980.8966 −115.52 0 (42)3.6 1 R.WPGGSISDVYNWETNTR.N 219 991.3461 1980.6777 1980.8966 −110.47 0(31) 40 1 R.WPGGSISDVYNWETNTR.N 221 991.8396 1981.6647 1981.8806 −108.910 85 0.00016 1 R.WPGGSISDVYNWETNTR.N + Glyc-Asn (N) 265 731.93922192.7957 2193.2117 −189.66 0 69 0.0086 1 R.GSNAAQILQTLQSISPLLSPR.A 2661097.4561 2192.8977 2193.2117 −143.15 0 (31) 56 1R.GSNAAQILQTLQSISPLLSPR.A 286 1133.4606 2264.9067 2265.1892 −124.70 0(31) 58 2 R.SPSTIYSADLNVLGVGPYAITK.A 287 1133.4786 2264.9427 2265.1892−108.81 0 (48) 0.95 1 R.SPSTIYSADLNVLGVGPYAITK.A 288 1133.4811 2264.94772265.1892 −106.60 0 70 0.0073 1 R.SPSTIYSADLNVLGVGPYAITK.A 289 756.15952265.4567 2265.1892 118 0 (21) 4.5e+02 9 R.SPSTIYSADLNVLGVGPYAITK.A 3091177.9986 2353.9827 2354.2481 −112.72 0 68 0.012 1K.ALVYGEGSSAVSPALTLPTAHSVK.L 310 1178.0016 2353.9887 2354.2481 −110.17 0(51) 0.59 1 K.ALVYGEGSSAVSPALTLPTAHSVK.L 311 1178.0086 2354.00272354.2481 −104.22 0 (54) 0.26 1 K.ALVYGEGSSAVSPALTLPTAHSVK.L 3191182.9381 2363.8617 2364.1346 −115.41 0 (91) 5.1e−05 1R.TWSSFETQVDPQGAAQTALATR.I 320 1182.9386 2363.8627 2364.1346 −114.99 0(109)  7.9e−07 1 R.TWSSFETQVDPQGAAQTALATR.I 321 1182.9416 2363.86872364.1346 −112.45 0 (115)  2.1e−07 1 R.TWSSFETQVDPQGAAQTALATR.I 3221182.9781 2363.9417 2364.1346 −81.57 0 (83) 0.00036 1R.TWSSFETQVDPQGAAQTALATR.I 323 1182.9866 2363.9587 2364.1346 −74.38 0126  1.6e−08 1 R.TWSSFETQVDPQGAAQTALATR.I 324 789.1825 2364.52572364.1346 165 0 (17) 1.1e+03 9 R.TWSSFETQVDPQGAAQTALATR.I 307 1188.32812374.6417 2374.1851 192 0 88 7.6e−05 1 K.GNPGLSPQAYAQNALQFIQAMR.A 3321188.4176 2374.8207 2375.1691 −146.69 0 (72) 0.0042 1K.GNPGLSPQAYAQNALQFIQAMR.A + Glyc-Asn (N) 333 1188.4181 2374.82172375.1691 −146.26 0 (76) 0.0019 1 K.GNPGLSPQAYAQNALQFIQAMR.A + Glyc-Asn(N) 337 1188.9406 2375.8667 2376.1531 −120.54 0 (66) 0.017 1K.GNPGLSPQAYAQNALQFIQAMR.A + 2 Glyc-Asn (N) 397 1288.5416 2575.06872575.3605 −113.30 0 (130)  8.2e−09 1 R.QASSSIVGNALAQAASLSPTISAYLR.Q 3981288.5621 2575.1097 2575.3605 −97.38 0 135  2.6e−09 1R.QASSSIVGNALAQAASLSPTISAYLR.Q 399 859.5002 2575.4787 2575.3605 45.9 0(64) 0.034 1 R.QASSSIVGNALAQAASLSPTISAYLR.Q 400 859.8579 2576.55172576.3445 80.4 0 (74) 0.0035 1 R.QASSSIVGNALAQAASLSPTISAYLR.Q + Glyc-Asn(N)

Example 4 Glycoprotein Staining of Proteins from Alicyclobacillusacidocaldarius

Alicyclobacillus acidocaldarius was cultured on wheat arabinoxylan andharvested after three days. The culture was centrifuged to remove cellsand the resulting supernatant was filtered with a 0.22 micron filter toremove any remaining debris. The filtered supernatant was concentratedto approximately 1 mL by ultrafiltration through a 10,000 Da molecularweight cutoff membrane. Several lanes of this concentrated material wererun on a 12% SDS-PAGE gel along with a positive and negative controlthat are known glycosylated and non-glycosylated proteins using standardprotocols. The gel was cut in half vertically and one half stained usingSIMPLY BLUE™ SAFE STAIN and the other half using a glycoproteindetection kit from Sigma. The positive and negative controls bothstained using the SIMPLY BLUE™ SAFE STAIN and only the positive controlstained with the glycoprotein stain indicating that the stainingprotocol was working correctly. The Alicyclobacillus acidocaldariusprotein lanes revealed a band at approximately 120 kDa on the SIMPLYBLUE™ stained gel which is the expected weight of one extracelluarprotein of Alicyclobacillus acidocaldarius. The same position on theglycoprotein stained gel showed pink bands indicating a positive resultfor a glycosylated protein.

Example 5 Chemical Glycosylation of SEQ ID NO:307 Results in GreaterXylanase Activity

SEQ ID NO:307 was expressed in E. coli and purified using a Co-resinsystem. The nucleic acid encoding SEQ ID NO:307 was altered for optimalcodon usage in E. coli. The purified SEQ ID NO:307 was chemicallyglycosylated using a mono(lactosylamido) mono(succinimidyl)suberate.This chemically reacts with amine groups on proteins to form an N-linkedmodification with a terminal lactose on the protein The glycosylation ofthe purified SEQ ID NO:307 was verified using a glycosylation stain.Purified glycosylated and un-glycosylated SEQ ID NO:307 was tested forxylanase activity at pHs 2, 3.5, and 5. The glycosylated SEQ ID NO:307had higher levels of activity at pH 2 and 3.5 that the ungylcosylatedfrom of SEQ ID NO:307. Un-glycosylated SEQ ID NO:307 displayed reducedsolubility at pH 2 and 3.5.

Example 6 Expression of SEQ ID NO:307 in Pichia pastoris

Nucleic acid encoding SEQ ID NO:307 was inserted into Pichia pastorisand several clones had significant xylanase and cellulose activities atpH 3.5, but not at pH 2. The nucleic acid encoding SEQ ID NO:307 wasaltered for optimal codon usage in Pichia pastoris.

Example 7 Comparison of Xylanase and Cellulase Activity of SEQ ID NO:307Expressed in Alicyclobacillus acidocaldarius and E. coli.

Nucleic acid encoding SEQ ID NO:307 was inserted into E. coli with anN-terminal His tag and the resultant protein purified. The nucleic acidencoding SEQ ID NO:307 was altered for optimal codon usage in E. coli.The resulting purified protein had no glycosylation. Protein of SEQ IDNO:307 was also purified from Alicyclobacillus acidocaldarius. Thepurified protein from Alicyclobacillus acidocaldarius was glycosylatedas normal for protein produced from this organism. The purified proteinswere tested for xylanase activity using wheat arabinoxylan (WAX). Theresults of the comparison are presented in FIG. 23. There was no dataavailable for enzyme purified from Alicyclobacillus acidocaldarius at pH5.5. As can be seen in FIG. 23, the Alicyclobacillus acidocaldariuspurified enzyme (black bars) had significantly more xylanase activity atpH 2 and 80° C. than the E. coli purified enzyme (white bars).

The purified proteins were also tested for cellulase activity usingcarboxymethyl cellulose (CMC). The results of the comparison arepresented in FIG. 24. There was no data available for enzyme purifiedfrom Alicyclobacillus acidocaldarius at pH 5.5. As can be seen in FIG.24, the Alicyclobacillus acidocaldarius purified enzyme (black bars) hadsignificantly less cellulase activity at pH 3.5 than the E. colipurified enzyme (white bars).

FIG. 25 presents the ratio of cellulose/xylanase activity for the datapresented in FIGS. 23 and 24. As can be seen therein, enzyme purifiedfrom Alicyclobacillus acidocaldarius had predominantly xylanase activityat pH 2 and 80° C., while having predominantly cellulose activity at allother conditions tested (black bars). The enzyme purified from E. colihad predominantly cellulose activity at all conditions tested (whitebars). This data confirms that the glycosylation state of SEQ ID NO:307varies the relative xylanase and cellulose activities of SEQ ID NO:307.

Example 8 Comparison of Xylanase and Cellulase Activity of SEQ ID NO:307Expressed in E. coli and Pichia pastoris.

Nucleic acid encoding SEQ ID NO:307 was inserted into Pichia pastorisand the resultant protein purified. The nucleic acid encoding SEQ IDNO:307 was altered for optimal codon usage in Pichia pastoris. Thepurified protein from Pichia pastoris was glycosylated as normal forprotein produced from this organism. Nucleic acid encoding SEQ ID NO:307was inserted into E. coli with an N-terminal His tag and the resultantprotein purified. The nucleic acid encoding SEQ ID NO:307 was alteredfor optimal codon usage in E. coli. The resulting purified protein hadno glycosylation. The purified proteins were tested for xylanaseactivity using wheat arabinoxylan. The results of the comparison arepresented in FIG. 26. As can be seen in FIG. 26, the Pichia pastorispurified enzyme (black bars) had significantly more xylanase activity atall conditions other than pH 3.5 and 80° C. than the E. coli purifiedenzyme (white bars).

The purified proteins were also tested for cellulase activity usingcarboxymethyl cellulose. The results of the comparison are presented inFIG. 27. As can be seen in FIG. 27, the Pichia pastoris purified enzyme(black bars) had significantly greater cellulase activity at 60° C.while the E. coli purified enzyme (white bars) displayed significantlygreater cellulase activity at 80° C.

FIG. 28 presents the ratio of cellulose/xylanase activity for the datapresented in FIGS. 26 and 27. As can be seen therein, enzyme purifiedfrom Pichia pastoris had predominantly cellulase activity at allconditions other than pH 2 and 80° C. The enzyme purified from E. colihad predominantly cellulose activity at all conditions tested. This dataconfirms that the glycosylation state of SEQ ID NO:307 varies therelative xylanase and cellulose activities of SEQ ID NO:307.

Example 9 Comparison of Xylanase and Cellulase Activity of SEQ ID NO:307Expressed in Alicyclobacillus acidocaldarius and Pichia pastoris

Nucleic acid encoding SEQ ID NO:307 was inserted into Pichia pastorisand the resultant protein purified. The nucleic acid encoding SEQ IDNO:307 was altered for optimal codon usage in Pichia pastoris. Thepurified protein from Pichia pastoris was glycosylated as normal forprotein produced from this organism. Protein of SEQ ID NO:307 was alsopurified from Alicyclobacillus acidocaldarius. The purified protein fromAlicyclobacillus acidocaldarius was glycosylated as normal for proteinproduced from this organism. The purified proteins were tested forxylanase activity using wheat arabinoxylan. The results of thecomparison are presented in FIG. 29. There was no data available forenzyme purified from Alicyclobacillus acidocaldarius at pH 5.5. As canbe seen in FIG. 29, the Alicyclobacillus acidocaldarius purified enzyme(black bars) had significantly more xylanase activity at a pH of 2 and apH of 3.5 and 80° C. than the Pichia pastoris purified enzyme (whitebars).

The purified proteins were also tested for cellulase activity usingcarboxymethyl cellulose. The results of the comparison are presented inFIG. 30. There was no data available for enzyme purified fromAlicyclobacillus acidocaldarius at pH 5.5. As can be seen in FIG. 30,the Alicyclobacillus acidocaldarius purified enzyme (black bars) hadsignificantly less cellulase activity at all conditions other than pH3.5 and 80° C. than the Pichia pastoris purified enzyme (white bars).

FIG. 31 presents the ratio of cellulose/xylanase activity for the datapresented in FIGS. 29 and 30. As can be seen therein, enzyme purifiedfrom Alicyclobacillus acidocaldarius had predominantly xylanase activityat pH 2 and 80° C., while having predominantly cellulose activity at allother conditions tested (black bars). The enzyme purified from Pichiapastoris also had predominantly xylanase activity at pH 2 and 80° C.,while having predominantly cellulase activity at all other conditionstested (white bars). This data confirms that the glycosylation state ofSEQ ID NO:307 varies the relative xylanase and cellulose activities ofSEQ ID NO:307.

Example 10 Comparison of Xylanase and Cellulase Activity of TruncatedSEQ ID NO:307 Expressed in E. coli and Pichia pastoris.

Nucleic acid encoding a truncated SEQ ID NO:307 without the 203C-terminal amino acids was inserted into Pichia pastoris with aC-terminal His tag and the resultant protein purified. The nucleic acidencoding SEQ ID NO:307 was altered for optimal codon usage in Pichiapastoris. The purified protein from Pichia pastoris was glycosylated asnormal for protein produced from this organism. Nucleic acid encoding atruncated SEQ ID NO:307 without the 203 C-terminal amino acids andN-terminal 33 leader sequence amino acids were inserted into E. coliwith an and the resultant protein purified. The nucleic acid encodingSEQ ID NO:307 was altered for optimal codon usage in E. coli. Theresulting purified protein had no glycosylation. The purified proteinswere tested for xylanase activity using wheat arabinoxylan. The resultsof the comparison are presented in FIG. 32. As can be seen in FIG. 32,the Pichia pastoris purified enzyme (black bars) had significantly morexylanase activity at all 60° C. conditions while the E. coli purifiedenzyme (white bars) had more xylanase activity at all 80° C. conditions.

The purified proteins were also tested for cellulase activity usingcarboxymethyl cellulose. The results of the comparison are presented inFIG. 33. As can be seen in FIG. 33, the Pichia pastoris purified enzyme(black bars) had significantly greater cellulase activity 60° C. and pHs3.5 and 5.5 while the E. coli purified enzyme (white bars) displayedgreater cellulase at all other conditions.

FIG. 34 presents the ratio of cellulose/xylanase activity for the datapresented in FIGS. 32 and 33. As can be seen therein, both purifiedenzymes had predominantly cellulase activity at all conditions.

Example 11 Activity of SEQ ID NO:338 Expressed in E. coli and Pichiapastoris

RAAC00568 (SEQ ID NO:338) was expressed in both E. coli and Pichiapastoris. Codon usage in the encoding DNA was optimized for theparticular organism. In E. coli, the enzyme expressed primarily asinclusion bodies. A small amount of enzyme was soluble and we were ableto purify it via the His tag by immobilized metal affinitychromatography (IMAC). This purified enzyme was tested for both alphaglucosidase and alpha xylosidase activities at pH 6.0 and 60° C. and wasfound to have no activity. Several attempts were also made to solubilizethe inclusion bodies and did not result in active protein. In Pichiapastoris, a soluble enzyme was expressed and purified by IMAC. Thisenzyme had activity at pH 5.5 and 60° C. for both alpha glucosidase andalpha xylosidase. These results demonstrate that the expression systemand glycosylation state of RAAC00568 may alter both the solubility andactivity of the enzyme.

Example 12 Activity of SEQ ID NO:337 Expressed in E. coli and Pichiapastoris

RAAC00307 (SEQ ID NO:337) was expressed in both E. coli and Pichiapastoris. Codon usage in the encoding DNA was optimized for theparticular organism. The E. coli produced enzyme was purified via a Histag by immobilized metal affinity chromatography (IMAC). This purifiedenzyme was tested for both alpha arabinofuranosidase (AFS) activity, aswell as beta xylosidase (BXYL) activity. The results of this testing arepresented in FIGS. 35-37. In Pichia pastoris, a soluble enzyme wasexpressed and purified by IMAC.

The optimum conditions for the E. coli expressed AFS were pH 6.0 and 70°C., while the optimum conditions for BXYL were pH 5.0 and between 70° C.and 80° C. (FIGS. 35 and 36). The enzyme did not have activity at pH 2.0for either AFS or BXYL activities (FIGS. 35 and 36). The Pichia pastorisexpressed enzyme was screened at pH 2, 3.5, and 5.5 and at 60° C. and80° C. The results are presented in FIG. 37. The glycosylationmodifications made by Pichia pastoris during expression appeared to haveshifted the activity to a lower pH. The BXYL in the Pichia pastorisexpressed enzyme was 10.8 U/mg at pH 3.0, 60° C., while it was only 1.1U/mg for the E. coli expressed enzyme. The Pichia pastoris expressedenzyme also had some activity at pH 2.0 while there was no activity forthe E. coli expressed enzyme. These results demonstrate that theexpression system and glycosylation state of RAAC00307 may alter itsactivity.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to thesame extent as if each reference were individually and specificallyindicated to be incorporated by reference and were set forth in itsentirety herein.

While this invention has been described in the context of certainembodiments, the present invention can be further modified within thespirit and scope of this disclosure. This application is thereforeintended to cover any variations, uses, or adaptations of the inventionusing its general principles. Further, this application is intended tocover such departures from the present disclosure as come within knownor customary practice in the art to which this invention pertains andwhich fall within the limits of the appended claims and their legalequivalents.

BIBLIOGRAPHIC REFERENCES

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1. A method of altering enzymatic activity of an extremophilic enzyme,the method comprising: placing the extremophilic enzyme in fluid contactwith an isolated and/or purified enzyme or a chemical system capable ofglycosylating the extremophilic enzyme; and glycosylating theextremophilic enzyme.
 2. The method according to claim 1, wherein theisolated and/or purified enzyme capable of glycosylating theextremophilic enzyme is isolated and/or purified from an organism. 3.The method according to claim 2, wherein the organism is selected fromthe group consisting of Alicyclobacillus acidocaldarius and Pichiapastoris.
 4. The method according to claim 1, wherein the extremophilicenzyme is an extremophilic enzyme of a thermoacidophile.
 5. The methodaccording to claim 4, wherein the extremophilic enzyme is selected fromthe group consisting of SEQ ID NOS:307, 337, and
 338. 6. The methodaccording to claim 1, wherein the extremophilic enzyme has alteredcellulase, xylanase, or beta xylosidase activity compared to anon-glycosylated form of the extremophilic enzyme.
 7. The methodaccording to claim 1, wherein the glycosylated extremophilic enzyme hasaltered cellulase, xylanase, or beta xylosidase activity compared to anon-glycosylated form of the extremophilic enzyme at a pH selected fromthe group consisting of a pH of less than about 5, about 3.5, and about2 or at a temperature selected from the group consisting of less thanabout 90° C., about 90° C., about 80° C., about 70° C., about 60° C.,and about 50° C.
 8. A method of altering enzymatic activity of anextremophilic enzyme, the method comprising: placing the extremophilicenzyme in fluid contact with a cell extract of an organism capable ofglycosylating the extremophilic enzyme; and glycosylating theextremophilic enzyme.
 9. The method according to claim 8, wherein theorganism is selected from the group consisting of Alicyclobacillusacidocaldarius and Pichia pastoris.
 10. The method according to claim 8,wherein the extremophilic enzyme is an extremophilic enzyme of athermoacidophile.
 11. The method according to claim 10, wherein theextremophilic enzyme is selected from the group consisting of SEQNOS:307, 337, and
 338. 12. The method according to claim 8, wherein theglycosylated extremophilic enzyme has altered cellulase, xylanase, orbeta xylosidase activity compared to a non-glycosylated form of theextremophilic enzyme.
 13. The method according to claim 8, wherein theglycosylated extremophilic enzyme has altered cellulase, xylanase, orbeta xylosidase activity compared to a non-glycosylated form of theextremophilic enzyme at a pH selected from the group consisting of a pHof less than about 5, about 3.5, and about 2 or at a temperatureselected from the group consisting of less than about 90° C., about 90°C., about 80° C., about 70° C., about 60° C., and about 50° C.
 14. Amethod of altering enzymatic activity of an extremophilic enzyme, themethod comprising: providing a nucleic acid encoding the extremophilicenzyme to an organism capable of expressing and glycosylating theextremophilic enzyme; and producing a glycosylated form of theextremophilic enzyme from the organism.
 15. The method according toclaim 14, wherein the organism is selected from the group consisting ofAlicyclobacillus acidocaldarius and Pichia pastoris.
 16. The methodaccording to claim 14, wherein the extremophilic enzyme is an enzyme ofa thermoacidophile.
 17. The method according to claim 14, wherein theextremophilic enzyme is selected from the group consisting of SEQNOS:307, 337, and
 338. 18. The method according to claim 14, wherein thenucleic acid encoding the protein further comprises an excretion signal.19. The method according to claim 14, wherein the glycosylated form ofthe extremophilic enzyme has altered cellulase, xylanase, or betaxylosidase activity compared to a non-glycosylated form of theextremophilic enzyme.
 20. The method according to claim 14, wherein theglycosylated form of the extremophilic enzyme has altered cellulase,xylanase, or beta xylosidase activity compared to a non-glycosylatedform of the extremophilic enzyme at a pH selected from the groupconsisting of a pH of less than about 5, about 3.5, and about 2 or at atemperature selected from the group consisting of less than about 90°C., about 90° C., about 80° C., about 70° C., about 60° C., and about50° C.
 21. An isolated or purified extremophilic enzyme produced by anyone of the methods according to claims 1, 8, and
 14. 22. A method ofpost-translationally modifying a protein of interest, the methodcomprising: placing the protein of interest in fluid contact with aglycosyltransferase isolated from an organism capable of glycosylatingthe protein of interest.
 23. The method according to claim 22, whereinthe organism is selected from the group consisting of Alicyclobacillusacidocaldarius and Pichia pastoris.
 24. The method according to claim22, wherein the glycosyltransferase is selected from the groupconsisting of the glycosyltransferases of SEQ ID NOS:1, 18, 35, 52, 69,86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, and
 290. 25.The method according to claim 22, wherein the protein of interest is anextremophilic enzyme.
 26. The method according to claim 25, wherein theextremophilic enzyme is selected from the group consisting of SEQ IDNOS:307, 331, 333, 335, 337, and
 338. 27. The method according to claim22, wherein the post-translational modification comprises glycosylation.28. A method of post-translationally modifying a protein of interest,the method comprising: placing the protein of interest in fluid contactwith a cell extract of a thermoacidophilic organism.
 29. The methodaccording to claim 28, wherein the thermoacidophilic organism isselected from the group consisting of Alicyclobacillus acidocaldariusand Pichia pastoris.
 30. The method according to claim 28, wherein theprotein of interest is an enzyme of a thermoacidophile.
 31. The methodaccording to claim 30, wherein the protein of interest is selected fromthe group consisting of SEQ ID NOS:307, 331, 333, 335, 337 and
 338. 32.The method according to claim 28, wherein the post-translationalmodification comprises glycosylation.
 33. A method of glycosylating aprotein of interest, the method comprising: providing a nucleic acidencoding the protein of interest to an thermoacidophilic organism; andproducing the protein of interest in the thermoacidophilic organism. 34.The method according to claim 33, wherein the thermoacidophilic organismis selected from the group consisting of Alicyclobacillus acidocaldariusand Pichia pastoris.
 35. The method according to claim 33, wherein theprotein of interest is an endoglucanase and/or xylanase of athermoacidophile.
 36. The method according to claim 33, wherein theprotein of interest is selected from the group consisting of SEQ IDNOS:307, 331, 333, 335, 339, and
 340. 37. The method according to claim33, wherein the nucleic acid encoding the protein of interest furthercomprises an excretion signal.
 38. The method according to claim 33,wherein the post-translational modification comprises glycosylation. 39.An isolated and/or purified protein produced by any one of the methodsaccording to claims 22, 28, and
 33. 40. An isolated and/or purifiedglycosylated protein, wherein the protein is selected from the groupconsisting of SEQ ID NOS:307, 331, 333, 335, 337, and 338.